Development of enzyme-linked immunosorbent assays based on recombinant MSP1a and MSP2 of Anaplasma marginale

Indirect enzyme-linked immunosorbent assays (ELISAs) based on recombinant MSP1a and MSP2 from a Brazilian isolate of Anaplasma marginale were developed to detect antibodies against this rickettsia in cattle. The high sensitivities (99% for both tests) and specificities (100% for both tests) were confirmed with sera from cattle positive or negative for A. marginale antibodies, respectively, by immunofluorescent antibody test. By the analysis of 583 sera from cattle of three regions of the state of Pernambuco, Brazil, the agreement between both tests was high, with a kappa index of 0.89. The similar performances of the ELISAs suggest that both tests can be used in epidemiological surveys for detection of antibodies to A. marginale in cattle.

Polymerase chain reaction with lesion scrapping for the diagnosis of human American tegumentary leishmaniasis

The objective of this work was to compare the polymerase chain reaction (PCR) using lesion scrapping with other conventional techniques for the diagnosis of the American tegumentary leishmaniasis (ATL). For this, patients with cutaneous lesions suspected to be ATL were studied. The DNA was amplified with the MP1L/MP3H primers. From the 156 studied patients, 79 (50.6%) presented positive parasite direct search (PD), 81 (51.9%) had positive Montenegro skin test (MST), and 90 (57.7%) presented PD and/or MST positive. The PCR was positive in all of the positive-PD patients (100% sensitivity), in 91.1% of the positive PD and/or MST patients, and in 27.3% of the patients that presented negative PD and positive MST. The PCR positivity was similar to the PD (P = 0.2482) and inferior to the MST (P = 0.0455), and to the PD/MST association (P = 0.0133). The high PCR sensitivity, and positivity in those cases where the PD was negative, highlights the importance of this technique as an auxiliary tool for the diagnosis of ATL.

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.

Diagnosis of Streptococcus pneumoniae meningitis by polymerase chain reaction amplification of the gene for pneumolysin

Diagnosis of bacterial meningitis has long been based on classical methods of Gram stain, serological tests, and culture of cerebrospinal fluid (CSF). The performance of these methods, especially culture and direct smear, is thwarted by failure to detect bacteria following administration of antimicrobial agents and reluctance to performance lumbar punctures at admission. Indeed, patients with meningitis frequently receive antibiotics orally or by injection before the diagnosis is suspected or established. Thus an alternative method has become necessary to help clinicians and epidemiologists to management and control of bacterial meningitis. We evaluate the application of a polymerase chain reaction-based (PCR) assay for amplification of pneumolysin gene (ply) to diagnosis of Streptococcus pneumoniae meningitis. The PCR assay sensitivity for CSF was 96% (95% confidence interval, CI, 90-99%) compared to a sensitivity of 59% for culture (95% CI 49-69%), 66% for Gram stain (95% CI 56-74%), and 78% for latex agglutination test (95% CI 69-86%); PCR specificity was 100% (95% CI 83-100%). PCR results were available within 4 h of the start of the assay. This molecular approach proved to be reliable and useful to identify this bacterium compared with other classical laboratory methods for identification of bacterial meningitis pathogens.

Assessment of a two-step nucleic acid amplification assay for detection of Neisseria meningitidis followed by capsular genogrouping

Immediate prevention of meningococcal disease relies in part on the prompt treatment with antibiotics of household and other close contacts of cases; however intervention with effective vaccination relies on identification of serogroup-causing strains. Parenteral antibiotic for patient with suspected meningococcal disease before hospital admission is currently recommended. Laboratory standard methods are hindered by failure to detect bacteria by this medical approach to improve patient prognosis. We assessed two polymerase chain reaction (PCR) assays to detect (crgA) and define the serogroups (siaD, orf-2, and ctrA) of Neisseria meningitidis in 120 cerebrospinal fluid (CSF) samples from positive cases (culture or antigen detection or direct smear). The PCR sensitivity for the identification of N. meningitidis was 100% (95% confidence interval, CI, 96-100%) compared to a sensitivity of 46% for culture (95% CI 37-55%), 61% for latex agglutination test (95% CI 52-70%), and 68% for Gram stain (95% CI 59-76%); PCR specificity was 97% (95% CI 82-100%). PCR correctly identified the serogroups A, B, C, W135, Y, and X in CSF samples with a sensitivity of 88% (95% CI 80-93%); the primer sets were 100% specific. The introduction of PCR-based assays shall increase laboratory confirmed cases, consequently enhancing surveillance of meningococcal disease.

Evaluation of a malaria antibody enzyme immunoassay for use in blood screening

Transfusion-transmitted malaria is rare, but it may produce severe problem in the safety of blood transfusion due to the lack of reliable procedure to evaluate donors potentially exposed to malaria. Here, we evaluated a new enzyme-linked immunosorbent assay malaria antibody test (ELISA malaria antibody test, DiaMed, Switzerland) to detect antibodies to Plasmodium vivax (the indigenous malaria) in the blood samples in the Republic of Korea (ROK). Blood samples of four groups were obtained and analyzed; 100 samples from P.vivax infected patients, 35 from recovery patients, 366 from normal healthy individuals, and 325 from domestic travelers of non-endemic areas residents to risky areas of ROK. P.vivax antibody levels by ELISA were then compared to the results from microscopic examination and polymerase chain reaction (PCR) test. As a result, the ELISA malaria antibody test had a clinical sensitivity of 53.0% and a clinical specificity of 94.0% for P.vivax. Twenty out of 325 domestic travelers (6.2%) were reactive and 28 cases (8.6%) were doubtful. Of the reactive and doubtful cases, only two were confirmed as acute malaria by both microscopy and PCR test. Thus we found that the ELISA malaria antibody test was insufficiently sensitive for blood screening of P.vivax in ROK.

Survey to identify Mycobacterium leprae-infected household contacts of patients from prevalent regions of leprosy in Colombia

Leprosy in Colombia is in the post-elimination phase; nevertheless, there are regions of this country where the incidence is still around 3-4/100,000. Early detection of leprosy patients is a priority for achieving control and elimination of leprosy; however, the clinical exam is not very sensitive and thus, the majority of patients are diagnosed only when they demonstrate lesions, and damage to the nerves and skin has already occurred. The goal of the present study was to identify Mycobacterium leprae infection and immune responses in household contacts (HHC) of leprosy patients from three prevalent regions of Colombia. Clinical examination, the Mitsuda test, evaluation of IgM anti-PGL-I in the serum, the bacillar index (BI), and polymerase chain reaction (PCR) from nasal swabs (NS) were performed for 402 HHC of 104 leprosy patients during a cross-sectional survey. Positive titers for IgM anti-PGL1 were found for 54 HHC, and PCR-positive NS for 22. The Mitsuda reaction was negative for 38 HHC, although three were positive for IgM anti-PGL-1 titers. The data document that leprosy transmission among HHC is still occurring in a non-endemic country.

Synthesis of limonene β-amino alcohol derivatives in support of new antileishmanial therapies

A series of seven limonene β-amino alcohol derivatives has been regioselectively synthesised in moderate to good yields. Two of these compounds were found to be significantly effective against in vitro cultures of the Leishmania (Viannia) braziliensis promastigote form in the micromolar range. The activities found for 3b and 3f were about 100-fold more potent than the standard drug, Pentamidine, in the same test, while limonene did not display any activity. This is the first report of antileishmanial activity by limonene β-amino alcohol derivatives.

Effectiveness and safety of isoniazid chemoprophylaxis for HIV-1 infected patients from Rio de Janeiro

The clinical and epidemiological characteristics, adverse events, treatment adherence and effectiveness of isoniazid chemoprophylaxis were analyzed in a cohort of 138 tuberculosis/HIV-coinfected patients. An open, non-randomized, pragmatic prophylactic trial was conducted on adult patients with a normal chest X-ray and positive tuberculin skin test (> 5 mm) who received isoniazid chemoprophylaxis (300 mg/day) for six months. The mean of follow up was 2.8 years (SD 1.3). Adherence to chemoprophylaxis was 87.7% (121/138). Only one patient presented tuberculosis after the end of chemoprophylaxis, corresponding to 0.3 cases per 100 persons per year. The relative risk of some adverse effects was 4.6 times higher (95% CI: 1.9-11.5) in patients with positive anti-HCV serology (4/9, 44.4%) compared to those with negative serology (12/129, 9.6%) (p = 0.002). This study provides evidence regarding the effectiveness and safety of a short and self-administered isoniazid regimen. We recommend the implementation of this routine by health service practitioners.

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.