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.

Comparison between splenic and lymph node aspirations as sampling methods for the parasitological detection of Leishmania chagasi infection in dogs

The sensitivities of spleen and lymph node cultures for the diagnosis of canine visceral leishmaniasis were compared in 64 anti-Leishmania antibody positive dogs from an endemic area in Brazil. The sensitivity of spleen cultures for Leishmania detection was 97.9%; in lymph node cultures it was 25%. Positive spleen culture was more frequent (p = 0.048, Fisher's exact probability test) in symptomatic (28 out of 33 animals) than in asymptomatic animals (19 out of 31 animals). These results support the use of spleen instead of lymph node aspiration as the choice method for the parasitological diagnosis of the infection.

Comparison of methods for the identification of coagulase-negative staphylococci

Coagulase-negative staphylococci (CNS) species identification is still difficult for most clinical laboratories. The scheme proposed by Kloos and Schleifer and modified by Bannerman is the reference method used for the identification of staphylococcal species and subspecies; however, this method is relatively laborious for routine use since it requires the utilization of a large number of biochemical tests. The objective of the present study was to compare four methods, i.e., the reference method, the API Staph system (bioMérieux) and two methods modified from the reference method in our laboratory (simplified method and disk method), in the identification of 100 CNS strains. Compared to the reference method, the simplified method and disk method correctly identified 100 and 99% of the CNS species, respectively, while this rate was 84% for the API Staph system. Inaccurate identification by the API Staph method was observed for Staphylococcus epidermidis (2.2%), S. hominis (25%), S. haemolyticus (37.5%), and S. warneri (47.1%). The simplified method using the simple identification scheme proposed in the present study was found to be efficient for all strains tested, with 100% sensitivity and specificity and proved to be available alternative for the identification of staphylococci, offering, higher reliability and lower cost than the currently available commercial systems. This method would be very useful in clinical microbiology laboratory, especially in places with limited resources.

Evaluation of rapid alternative methods for drug susceptibility testing in clinical isolates of Mycobacterium tuberculosis

A study was carried out to compare the performance of a commercial method (MGIT) and four inexpensive drug susceptibility methods: nitrate reductase assay (NRA), microscopic observation drug susceptibility (MODS) assay, MTT test, and broth microdilution method (BMM). A total of 64 clinical isolates of Mycobacterium tuberculosis were studied. The Lowenstein-Jensen proportion method (PM) was used as gold standard. MGIT, NRA, MODS, and MTT results were available on an average of less than 10 days, whereas BMM results could be reported in about 20 days. Most of the evaluated tests showed excellent performance for isoniazid and rifampicin, with sensitivity and specificity values > 90%. With most of the assays, sensitivity for ethambutol was low (62-87%) whereas for streptomycin, sensitivity values ranged from 84 to 100%; NRA-discrepancies were associated with cultures with a low proportion of EMB-resistant organisms while most discrepancies with quantitative tests (MMT and BMM) were seen with isolates whose minimal inhibitory concentrations fell close the cutoff. MGIT is reliable but still expensive. NRA is the most inexpensive and easiest method to perform without changing the organization of the routine PM laboratory performance. While MODS, MTT, and BMM, have the disadvantage from the point of view of biosafety, they offer the possibility of detecting partial resistant strains. This study shows a very good level of agreement of the four low-cost methods compared to the PM for rapid detection of isoniazid, rifampicin and streptomycin resistance (Kappa values > 0.8); more standardization is needed for ethambutol.

Detection of rifampin resistance patterns in Mycobacterium tuberculosis strains isolated in Iran by polymerase chain reaction-single-strand conformation polymorphism and direct sequencing methods

Mutations in the rpoB locus confer conformational changes leading to defective binding of rifampin (RIF) to rpoB and consequently resistance in Mycobacterium tuberculosis. Polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) was established as a rapid screening test for the detection of mutations in the rpoB gene, and direct sequencing has been unambiguously applied to characterize mutations. A total of 37 of Iranian isolates of M. tuberculosis, 16 sensitive and 21 resistant to RIF, were used in this study. A 193-bp region of the rpoB gene was amplified and PCR-SSCP patterns were determined by electrophoresis in 10% acrylamide gel and silver staining. Also, 21 samples of 193-bp rpoB amplicons with different PCR-SSCP patterns from RIFr and 10 from RIFs were sequenced. Seven distinguishable PCR-SSCP patterns were recognized in the 21 Iranian RIFr strains, while 15 out of 16 RIFs isolates demonstrated PCR-SSCP banding patterns similar to that of sensitive standard strain H37Rv. However one of the sensitive isolates demonstrated a different pattern. There were seen six different mutations in the amplified region of rpoB gene: codon 516(GAC/GTC), 523(GGG/GGT), 526(CAC/TAC), 531(TCG/TTG), 511(CTG/TTG), and 512(AGC/TCG). This study demonstrated the high specificity (93.8%) and sensitivity (95.2%) of PCR-SSCP method for detection of mutation in rpoB gene; 85.7% of RIFr strains showed a single mutation and 14.3% had no mutations. Three strains showed mutations caused polymorphism. Our data support the common notion that rifampin resistance genotypes are generally present mutations in codons 531 and 526, most frequently found in M. tuberculosis populations regardless of geographic origin.

The impact of two education methods on knowledge of schistosomiasis transmission and prevention among schoolchildren in a rural community in northern Minas Gerais, Brazil

The objective of this study was to analyse the effect of using two health education approaches on knowledge of transmission and prevention of schistosomiasis of school children living in a rural endemic area in the state of Minas Gerais, Brazil. The 87 children participating in the study were divided into three groups based on gender, age and presence or absence of Schistosoma mansoni infection. In the first group the social representation model and illness experience was used. In the second group, we used the cognitive model based on the transmission of information. The third group, the control group, did not receive any information related to schistosomiasis. Ten meetings were held with all three groups that received a pre-test prior to the beginning of the educational intervention and a post-test after the completion of the program. The results showed that knowledge levels in Group 1 increased significantly during the program in regard to transmission (p = 0.038) and prevention (p = 0.001) of schistosomiasis. Groups 2 and 3 did not show significant increase in knowledge between the two tests. These results indicate that health education models need to consider social representation and illness experience besides scientific knowledge in order to increase knowledge of schistosomiasis transmission and prevention.

A closer look at multiple-clone Plasmodium vivax infections: detection methods, prevalence and consequences

The naturally occurring clonal diversity among field isolates of the major human malaria parasite Plasmodium vivax remained unexplored until the early 1990s, when improved molecular methods allowed the use of blood samples obtained directly from patients, without prior in vitro culture, for genotyping purposes. Here we briefly review the molecular strategies currently used to detect genetically distinct clones in patient-derived P. vivax samples, present evidence that multiple-clone P. vivax infections are commonly detected in areas with different levels of malaria transmission and discuss possible evolutionary and epidemiological consequences of the competition between genetically distinct clones in natural human infections. We suggest that, when two or more genetically distinct clones are present in the same host, intra-host competition for limited resources may select for P. vivax traits that represent major public health challenges, such as increased virulence, increased transmissibility and antimalarial drug resistance.

Diagnosis of congenital toxoplasmosis: prenatal and neonatal evaluation of methods used in Toulouse University Hospital and incidence of congenital toxoplasmosis

The aim of this study was to determine the incidence of congenital toxoplasmosis (CT) and to assess the performances of prenatal and neonatal diagnoses. From 1994-2005, in Toulouse University Hospital, France, amniocentesis was performed on 352 pregnant women who were infected during pregnancy. All women were treated with spiramycin and pyrimethamine-sulfadoxine when prenatal diagnosis was positive. Among the 275 foetuses with follow-up, 66 (24%) were infected. The transmission rates of Toxoplasma gondii were 7%, 24% and 59% in the first, second and third trimesters, respectively. The sensitivity and specificity of PCR on amniotic fluid (AF) were 91% and 99.5%, respectively. One case was diagnosed by mouse inoculation with AF and six cases were diagnosed by neonatal or postnatal screening. The sensitivity and specificity of PCR on placentas were 52% and 99%, respectively. The sensitivity of tests for the detection of specific IgA and IgM in cord blood was 53% and 64%, respectively, and specificity values were 91% and 92%. In conclusion, PCR performed on AF had the highest levels of sensitivity and specificity for the diagnosis of CT. This permits an early diagnosis of most cases and should be recommended.

Congenital toxoplasmosis: evaluation of serological methods for the detection of anti-Toxoplasma gondii IgM and IgA antibodies

A study was carried out to evaluate the presence of serological markers for the immunodiagnosis of the vertical transmission of toxoplasmosis. We tested the sensitivity, specificity and predictive values (positive and negative) of different serological methods for the early diagnosis of congenital toxoplasmosis. In a prospective longitudinal study, 50 infants with suspected congenital toxoplasmosis were followed up in the ambulatory care centre of Congenital Infections at University Hospital in Goiânia, Goiás, Brazil, from 1 January 2004-30 September 2005. Microparticle Enzyme Immunoassay (MEIA), Enzyme-Linked Fluorescent Assay (ELFA) and Immune-Fluorescent Antibody Technique (IFAT) were used to detect specific IgM anti-Toxoplasma gondii antibodies and a capture ELISA was used to detect specific IgA antibodies. The results showed that 28/50 infants were infected. During the neonatal period, IgM was detected in 39.3% (11/28) of those infected infants and IgA was detected in 21.4% (6/28). The sensitivity, specificity and predictive values (positive and negative) of each assay were, respectively: MEIA and ELFA: 60.9%, 100%, 100%, 55.0%; IFAT: 59.6%, 91.7%, 93.3%, 53.7%; IgA capture ELISA: 57.1%, 100%, 100%, 51.2%. The presence of specific IgM and IgA antibodies during the neonatal period was not frequent, although it was correlated with the most severe cases of congenital transmission. The results indicate that the absence of congenital disease markers (IgM and IgA) in newborns, even after confirming the absence with several techniques, does not constitute an exclusion criterion for toxoplasmosis.

Comparative study of two extraction methods for enteric virus recovery from sewage sludge by molecular methods

The aim of this study was to compare two nucleic acid extraction methods for the recovery of enteric viruses from activated sludge. Test samples were inoculated with human adenovirus (AdV), hepatitis A virus (HAV), poliovirus (PV) and rotavirus (RV) and were then processed by an adsorption-elution-precipitation method. Two extraction methods were used: an organic solvent-based method and a silica method. The organic-based method was able to recoup 20% of the AdV, 90% of the RV and 100% of both the PV and HAV from seeded samples. The silica method was able to recoup 1.8% of the AdV and 90% of the RV. These results indicate that the organic-based method is more suitable for detecting viruses in sewage sludge.

Accuracy of phenotypic methicillin susceptibility methods in the detection of Staphylococcus aureus isolates carrying different SCCmec types

A total of 138 isolates, 118 methicillin-resistant Staphylococcus aureus (MRSA) isolates (staphylococcal cassette chromosome type II, 20 isolates, type III, 39 isolates and type IV, 59 isolates) and 20 methicillin-sensitive S. aureus isolates were evaluated by phenotypic methods: cefoxitin and oxacillin disk diffusion (DD), agar dilution (AD), latex agglutination (LA), oxacillin agar screening (OAS) and chromogenic agar detection. All methods showed 100% specificity, but only the DD tests presented 100% sensitivity. The sensitivity of the other tests ranged from 82.2% (OAS)-98.3% (AD). The LA test showed the second lowest sensitivity (86.4%). The DD test showed high accuracy in the detection of MRSA isolates, but there was low precision in the detection of type IV isolates by the other tests, indicating that the genotypic characteristics of the isolates should be considered.

A comparative study of the TF-Test®, Kato-Katz, Hoffman-Pons-Janer, Willis and Baermann-Moraes coprologic methods for the detection of human parasitosis

This study compares the diagnostic accuracy of the TF-Test® (TFT) for human parasitosis with results obtained using the traditional Kato-Katz (KK), Hoffman-Pons-Janer (HPJ), Willis and Baermann-Moraes (BM) techniques. Overall, four stool samples were taken from each individual; three alternate-day TFT stool samples and another sample that was collected in a universal container. Stool samples were taken from 331 inhabitants of the community of Quilombola Santa Cruz. The gold standard (GS) for protozoa detection was defined as the combined results for TFT, HPJ and Willis coproscopic techniques; for helminth detection, GS was defined as the combined results for all five coproscopic techniques (TFT, KK, HPJ, Willis and BM). The positivity rate of each method was compared using the McNemar test. While the TFT exhibited similar positivity rates to the GS for Entamoeba histolytica/dispar (82.4%) and Giardia duodenalis (90%), HPJ and Willis techniques exhibited significantly lower positivity rates for these protozoa. All tests exhibited significantly lower positivity rates compared with GS for the diagnosis of helminths. The KK technique had the highest positivity rate for diagnosing Schistosoma mansoni (74.6%), while the TFT had the highest positivity rates for Ascaris lumbricoides (58.1%) and hookworm (75%); HPJ technique had the highest positivity rate for Strongyloides stercoralis (50%). Although a combination of tests is the most accurate method for the diagnosis of enteral parasites, the TFT reliably estimates the prevalence of protozoa and selected helminths, such as A. lumbricoides and hookworm. Further studies are needed to evaluate the detection accuracy of the TFT in samples with varying numbers of parasites.

Comparison of two PCR methods for detection of Leptospira interrogans in formalin-fixed and paraffin-embedded tissues

In this study we compared two polymerase chain reaction (PCR) methods using either 16S ribosomal RNA (rRNA) or 23S rRNA gene primers for the detection of different Leptospira interrogans serovars. The performance of these two methods was assessed using DNA extracted from bovine tissues previously inoculated with several bacterial suspensions. PCR was performed on the same tissues before and after the formalin-fixed, paraffin-embedding procedure (FFPE tissues). The 23S rDNA PCR detected all fresh and FFPE positive tissues while the 16S rDNA-based protocol detected primarily the positive fresh tissues. Both methods are specific for pathogenic L. interrogans. The 23S-based PCR method successfully detected Leptospira in four dubious cases of human leptospirosis from archival tissue specimens and one leptospirosis-positive canine specimen. A sensitive method for leptospirosis identification in FFPE tissues would be a useful tool to screen histological specimen archives and gain a better assessment of human leptospirosis prevalence, especially in tropical countries, where large outbreaks can occur following the rainy season.