Search

Over the past ten years, standard diagnostics for cryptococcal meningitis in HIV-infected persons have evolved from culture to India ink to detection of cryptococcal antigen (CrAg), with the recent development and distribution of a point-of-care lateral flow assay. This assay is highly sensitive and specific in cerebrospinal fluid (CSF), but is also sensitive in the blood to detect CrAg prior to meningitis symptoms. CrAg screening of HIV-infected persons in the blood prior to development of fulminant meningitis and preemptive treatment for CrAg-positive persons are recommended by the World Health Organization and many national HIV guidelines. Thus, CrAg testing is occurring more widely, especially in resource-limited laboratory settings. CrAg titer predicts meningitis and death and could be used in the future to customize therapy according to burden of infection., (Copyright © 2019 American Society for Microbiology.)

The only currently commercialized point-of-care assay for tuberculosis (TB) that measures lipoarabinomannan (LAM) in urine (Alere LF-LAM) has insufficient sensitivity. We evaluated the potential of 100 novel monoclonal antibody pairs targeting a variety of LAM epitopes on a sensitive electrochemiluminescence platform to improve the diagnostic accuracy. In the screening, many antibody pairs showed high reactivity to purified LAM but performed poorly at detecting urinary LAM in clinical samples, suggesting differences in antigen structure and immunoreactivity of the different LAM sources. The 12 best antibody pairs from the screening were tested in a retrospective case-control study with urine samples from 75 adults with presumptive TB. The best antibody pair reached femtomolar analytical sensitivity for LAM detection and an overall clinical sensitivity of 93% (confidence interval [CI], 80% to 97%) and specificity of 97% (CI, 85% to 100%). Importantly, in HIV-negative subjects positive for TB by sputum smear microscopy, the test achieved a sensitivity of 80% (CI, 55% to 93%). This compares to an overall sensitivity of 33% (CI, 20% to 48%) of the Alere LF-LAM and a sensitivity of 13% (CI, 4% to 38%) in HIV-negative subjects in the same sample set. The capture antibody targets a unique 5-methylthio-d-xylofuranose (MTX)-dependent epitope in LAM that is specific to the Mycobacterium tuberculosis complex and shows no cross-reactivity with fast-growing mycobacteria or other bacteria. The present study provides evidence that improved assay methods and reagents lead to increased diagnostic accuracy. The results of this work have informed the development of a sensitive and specific novel LAM point-of-care assay with the aim to meet the WHO's performance target for TB diagnosis., (Copyright © 2018 Sigal et al.)

More sensitive tests are needed for the diagnosis of smear-negative and HIV-associated tuberculosis. This study compares the sensitivities and specificities of three molecular tests, namely, the Xpert MTB/RIF test, the Xpert Ultra (Ultra), and RealTi m e MTB (RT-MTB), in a high HIV prevalence setting. Symptomatic adults were recruited from three outpatient sites, and each provided 4 sputum specimens. The diagnostic performance of Xpert MTB/RIF, Ultra, and RT-MTB was evaluated, with culture as a reference standard. HIV infection occurred in 62% of patients, with a median CD4 count of 220 cells/µl. The Ultra test had the highest sensitivity of 89.3% (95% confidence interval [CI], 78.1 to 96) compared to those of the Xpert MTB/RIF at 82.1% (95% CI, 69.6 to 91.1; P  = 0.12) and RT-MTB at 78.6% (95% CI, 65.6 to 88.4; P  = 0.68). The specificity was highest with the Xpert MTB/RIF at 100% (95% CI, 98 to 100), followed by RealTi m e MTB at 96.7% (95% CI, 92.9 to 98.8; P  = 0.03) and the Ultra at 95.6% (95% CI, 91.5 to 98.1; P  = 0.08). In patients with smear-negative disease, the Ultra was more sensitive than the Xpert MTB/RIF (64.7% [95% CI, 38.3 to 85.8] versus 41.2% [95% CI, 18.4 to 67.1], respectively; P  = 0.12), and RT-MTB performed equally to Xpert MTB/RIF. In a comparison of the Ultra and RT-MTB on the same sputum specimen pellets, the Ultra was more sensitive than RT-MTB in the overall cohort (88.9% [95% CI, 77.4 to 95.8] versus 77.8% [95% CI, 64.4 to 88], respectively; P  = 0.03) and among people with HIV (87.5% [95% CI, 71 to 96.5] versus 68.6% [95% CI, 50 to 83.9], respectively; P  = 0.03). Although these results did not reach statistical significance, they suggest that the Ultra is more sensitive than the Xpert MTB/RIF and RT-MTB, most prominently in smear-negative disease. This was accompanied by a loss of specificity., (Copyright © 2018 American Society for Microbiology.)

The usefulness of an automated latex turbidimetric rapid plasma reagin (RPR) assay, compared to the conventional manual card test (serial 2-fold dilution method), for the diagnosis of syphilis and evaluation of treatment response remains unknown. We conducted (i) a cross-sectional study and (ii) a prospective cohort study to elucidate the correlation between automated and manual tests and whether a 4-fold decrement is a feasible criterion for successful treatment with the automated test, respectively, in HIV-infected patients, from October 2015 to November 2017. Study i included 518 patients. The results showed strong correlation between the two tests ( r = 0.931; P < 0.001). With a manual test titer of ≥1:8 plus a positive Treponema pallidum particle agglutination (TPPA) test as the reference standard for diagnosis, the optimal cutoff value for the automated test was 6.0 RPR units (area under the curve [AUC], 0.998), with positive predictive value (PPV) of 92.5% and negative predictive value (NPV) of 99.4%. Study ii enrolled 66 men with syphilis. Their RPR values were followed up until after 12 months of treatment. At 12 months, 77.3% and 78.8% of the patients achieved a 4-fold decrement in RPR titer by the automated and manual test, respectively. The optimal decrement rate in RPR titer by the automated test for a 4-fold decrement by manual card test was 76.54% (AUC, 0.96) (PPV, 96.1%; NPV, 80.0%). The automated RPR test is a good alternative to the manual test for the diagnosis of syphilis and evaluation of treatment response and is more rapid and can handle more specimens than the manual test without interpersonal variation in interpretation., (Copyright © 2018 American Society for Microbiology.)

The protozoan parasite Toxoplasma gondii causes severe opportunistic infections. Here, we report an unexpected diagnosis of cerebral toxoplasmosis. T. gondii was diagnosed by 16S and D2 large-subunit (LSU) ribosomal DNA (rDNA) sequencing of a cerebral biopsy specimen and confirmed by T. gondii-specific PCR and immunohistochemistry. The patient was later diagnosed with HIV/AIDS., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

A molecular diagnostic technique based on real-time PCR was developed for the simultaneous detection of three of the most frequent causative agents of fungal opportunistic pneumonia in AIDS patients: Pneumocystis jirovecii, Histoplasma capsulatum, and Cryptococcus neoformans/Cryptococcus gattii. This technique was tested in cultured strains and in clinical samples from HIV-positive patients. The methodology used involved species-specific molecular beacon probes targeted to the internal transcribed spacer regions of the rDNA. An internal control was also included in each assay. The multiplex real-time PCR assay was tested in 24 clinical strains and 43 clinical samples from AIDS patients with proven fungal infection. The technique developed showed high reproducibility (r(2) of >0.98) and specificity (100%). For H. capsulatum and Cryptococcus spp., the detection limits of the method were 20 and 2 fg of genomic DNA/20 μl reaction mixture, respectively, while for P. jirovecii the detection limit was 2.92 log10 copies/20 μl reaction mixture. The sensitivity in vitro was 100% for clinical strains and 90.7% for clinical samples. The assay was positive for 92.5% of the patients. For one of the patients with proven histoplasmosis, P. jirovecii was also detected in a bronchoalveolar lavage sample. No PCR inhibition was detected. This multiplex real-time PCR technique is fast, sensitive, and specific and may have clinical applications.

Molecular diagnostic tools have been used increasingly in the characterization of the transmission of cryptosporidiosis and microsporidiosis in developing countries. However, few studies have examined the distribution of Cryptosporidium species and Enterocytozoon bieneusi genotypes in AIDS patients receiving antiretroviral therapy. In the present study, 683 HIV-positive patients in the National Free Antiretroviral Therapy Program in China and 683 matched HIV-negative controls were enrolled. Cryptosporidium species and subtypes and Enterocytozoon bieneusi genotypes were detected and differentiated by PCR and DNA sequencing. The infection rates were 1.5% and 0.15% for Cryptosporidium and 5.7% and 4.2% for E. bieneusi in HIV-positive and HIV-negative participants, respectively. The majority (8/11) of Cryptosporidium cases were infections by zoonotic species, including Cryptosporidium meleagridis (5), Cryptosporidium parvum (2), and Cryptosporidium suis (1). Prevalent E. bieneusi genotypes detected, including EbpC (39), D (12), and type IV (7), were also potentially zoonotic. The common occurrence of EbpC was a feature of E. bieneusi transmission not seen in other areas. Contact with animals was a risk factor for both cryptosporidiosis and microsporidiosis. The results suggest that zoonotic transmission was significant in the epidemiology of both diseases in rural AIDS patients in China.

In this prospective, longitudinal study on 948 HIV-1-infected patients, subjects with an indeterminate IFN-γ (gamma interferon) release assay (IGRA) result at baseline were at significantly higher risk of developing AIDS-defining manifestations other than tuberculosis (TB) irrespective of CD4(+) T cell count. Thus, in HIV-1-infected patients with advanced quantitative CD4(+) T cell depletion, an indeterminate IGRA might indicate an additional loss of global T cell function, warranting detailed clinical evaluation and careful follow-up.

Diagnosis of opportunistic infections in HIV-infected individuals remains a major public health challenge, particularly in resource-limited settings. Here, we describe a rapid diagnostic system that delivers a panel of serologic immunoassay results using a single drop of blood, serum, or plasma. The system consists of disposable cartridges and a simple reader instrument, based on an innovative implementation of planar waveguide imaging technology. The cartridge incorporates a microarray of recombinant antigens and antibody controls in a fluidic channel, providing multiple parallel fluorescence immunoassay results for a single sample. This study demonstrates system performance by delivering antibody (Ab) reactivity results simultaneously for multiple antigens of HIV-1, Treponema pallidum (syphilis), and hepatitis C virus (HCV) in a collection of clinical serum, plasma, and whole-blood samples. By plotting antibody reactivity (fluorescence intensity) for known positive and negative samples, empirical reactivity cutoff values were defined. The HIV-1 assay shows 100% agreement with known seroreactivity for a collection of 82 HIV Ab-positive and 142 HIV Ab-negative samples, including multiple samples with HCV and syphilis coinfection. The treponema-specific syphilis assay correctly identifies 67 of 68 T. pallidum Ab-positive and 100 of 102 T. pallidum Ab-negative samples, and the HCV assay correctly identifies 59 of 60 HCV Ab-positive and 120 of 121 HCV Ab-negative samples. Multiplexed assay performance for whole-blood samples is also demonstrated. The ability to diagnose HIV and opportunistic infections simultaneously at the point of care should lead to more effective therapy decisions and improved linkage to care.

(1-3)-Beta-D-Glucan (BG) reactivity was tested in serum samples from 28 patients with human immunodeficiency virus infection or a hematological malignancy and Pneumocystis jirovecii pneumonia (PCP) and 28 control patients. The sensitivity and specificity of BG detection with the Fungitell assay for PCP were 100 and 96.4%, respectively, using a cutoff value of 100 pg/ml. Serum BG testing looks promising for the noninvasive diagnosis of PCP. Our data suggest that a higher cutoff value for the diagnosis of PCP than for the diagnosis of invasive aspergillosis or candidiasis could be used safely and will improve the specificity of the test.

Among 345 persons who underwent indirect hemagglutination (IHA) serological assays and assays of specific amebic antigens in their stool samples, 24 of 36 (66.7%) who were seropositive for Entamoeba histolytica had intestinal amebiasis as determined by antigen assays compared with 2 of 309 (0.2%) who were seronegative (odds ratio, 307; 95% confidence interval, 64.9 to 1,451). The estimated cost to detect a case of intestinal amebiasis by serology followed by antigen assays ($52) could be reduced by 74.3% and 69.9%, respectively, compared with the costs of the concurrent use of both assays ($202) and the antigen assays alone ($173). Our finding suggests that IHA assays followed by specific-amebic-antigen assays can be cost-effective in the diagnosis of intestinal amebiasis among persons with or without human immunodeficiency virus infection who are at risk for E. histolytica infection.

Nocardia asteroides was isolated after prolonged culture from the pericardial fluid of a human immunodeficiency virus-infected patient. The lengthy duration required for culture growth and identification of this N. asteroides isolate affected both initial therapeutic decisions and patient management. A proposed algorithm for the microbiological workup of pericardial fluid for possible Nocardia spp. is described in an effort to improve the timeliness of results.

In order to assess the applicability of multiplexed fluorescence in situ hybridization (FISH) assay for the clinical setting, we conducted retrospective analysis of 110 formalin-stored diarrheic stool samples from human immunodeficiency virus (HIV)/AIDS patients with intestinal microsporidiosis collected between 1992 and 2003. The multiplexed FISH assay identified microsporidian spores in 94 of 110 (85.5%) samples: 49 (52.1%) were positive for Enterocytozoon bieneusi, 43 (45.8%) were positive for Encephalitozoon intestinalis, 2 (2.1%) were positive for Encephalitozoon hellem, and 9 samples (9.6%) contained both E. bieneusi and E. intestinalis spores. Quantitative spore counts per ml of stool yielded concentration values from 3.5 x 10(3) to 4.4 x 10(5) for E. bieneusi (mean, 8.8 x 10(4)/ml), 2.3 x 10(2) to 7.8 x 10(4) (mean, 1.5 x 10(4)/ml) for E. intestinalis, and 1.8 x 10(2) to 3.6 x 10(2) for E. hellem (mean, 2.7 x 10(2)/ml). Identification of microsporidian spores by multiplex FISH assay was more sensitive than both Chromotrope-2R and CalcoFluor White M2R stains; 85.5% versus 72.7 and 70.9%, respectively. The study demonstrated that microsporidian coinfection in HIV/AIDS patients with intestinal microsporidiosis is not uncommon and that formalin-stored fecal samples older than 10 years may not be suitable for retrospective analysis by techniques targeting rRNA. Multiplexed FISH assay is a reliable, quantitative fluorescence microscopy method for the simultaneous identification of E. bieneusi, E. intestinalis, and E. hellem, as well as Encephalitozoon cuniculi, spores in fecal samples and is a useful tool for assessing spore shedding intensity in intestinal microsporidiosis. The method can be used for epidemiological investigations and applied in clinical settings.

Toxoplasma gondii is an important food- and waterborne opportunistic pathogen that causes severe disease in immunocompromised patients. T. gondii has an unusual clonal population structure consisting of three widespread lineages known as I, II, and III. To establish the genotypes of strains of T. gondii associated with human toxoplasmosis, we have developed a set of four highly sensitive and polymorphic nested PCR markers. Multiplex nested PCR analysis was used to genotype parasites in cerebral spinal fluid samples from 8 of 10 human immunodeficiency virus-positive patients. Remarkably, a majority of these patients had infections with type I strains or strains containing type I alleles, despite the fact that this lineage is normally uncommon in humans and animals. Multiplex analysis of these four unlinked makers was able to distinguish all three common genotypes and also detected two strains with mixed genotypes. Further analysis based on sequencing of a polymorphic intron revealed that one of these recombinant strains was an exotic lineage distinct from the archetypal clonal lineages. The multiplex nested PCR analysis described here will be useful for analyzing the contribution of parasite genotype to toxoplasmosis.

Human papillomavirus (HPV) has recently been associated with oral cancers. To prepare for a study of the natural history of oral HPV infection, the effect of the DNA purification method on HPV genomic DNA detection in Scope mouthwash oral rinse samples and the reproducibility of HPV detection in rinse samples collected 7 days apart were investigated. The study was conducted with a population at high risk for oral HPV infection: human immunodeficiency virus-infected men with CD4-cell counts <200. Five DNA purification methods were compared among equal aliquots of oral rinse samples collected from a subset of individuals. The purification methods included (i) proteinase K digestion (PKD) and heat inactivation; (ii) PKD and ethanol precipitation (EP); (iii) PKD, phenol-chloroform extraction, and EP; (iv) use of the Puregene DNA purification kit; and (v) use of the QIAamp DNA Blood Midi kit. HPV was detected by PCR amplification with PGMY09 and PGMY11 L1 primer pools and by use of a Roche linear array. Puregene-purified samples had higher human DNA yields and purities, and Puregene purification detected the greatest number of HPV-positive subjects and total HPV infections in comparison to the numbers detected by all other methods. The total number of HPV infections and HPV prevalence estimates were also higher for Puregene-processed oral rinse samples when a fixed volume (10 mul) rather than a fixed cell number ( approximately 50,000 cells) was used for PCR amplification. A good concordance was observed for oral HPV infection status (agreement, 80%; kappa value, = 0.60) and type-specific infection (agreement, 98%; kappa value, 0.57) in matched oral rinse samples. The method of DNA purification significantly affects the detection of HPV genomic DNA from oral rinse samples and may result in exposure misclassification that could contribute to the inconsistent associations reported in the literature.

Cerebral toxoplasmosis is the most common cerebral focal lesion in AIDS and still accounts for high morbidity and mortality in Brazil. Its occurrence is more frequent in patients with low CD4(+) T-cell counts. It is directly related to the prevalence of anti-Toxoplasma gondii antibodies in the population. Therefore, it is important to evaluate sensitive, less invasive, and rapid diagnostic tests. We evaluated the value of PCR using peripheral blood samples on the diagnosis of cerebral toxoplasmosis and whether its association with immunological assays can contribute to a timely diagnosis. We prospectively analyzed blood samples from 192 AIDS patients divided into two groups. The first group was composed of samples from 64 patients with cerebral toxoplasmosis diagnosed by clinical and radiological features. The second group was composed of samples from 128 patients with other opportunistic diseases. Blood collection from patients with cerebral toxoplasmosis was done before or on the third day of anti-toxoplasma therapy. PCR for T. gondii, indirect immunofluorescence, enzyme-linked immunosorbent assay, and an avidity test for toxoplasmosis were performed on all samples. The PCR sensitivity and specificity for diagnosis of cerebral toxoplasmosis in blood were 80% and 98%, respectively. Patients with cerebral toxoplasmosis (89%) presented higher titers of anti-T. gondii IgG antibodies than patients with other diseases (57%) (P<0.001). These findings suggest the clinical value of the use of both PCR and high titers of anti-T. gondii IgG antibodies for the diagnosis of cerebral toxoplasmosis. This strategy may prevent more invasive approaches.

Nocardia cyriacigeorgica is a recently characterized species within the genus of Nocardia. We report a brain abscess, following a primary pulmonary colonization, due to this species in a human immunodeficiency virus-infected patient. This case confirms that isolation of Nocardia in sputum is associated with a high risk of disseminated infection in immunocompromised patients.

Highly active antiretroviral therapy has decreased the incidence of opportunistic infections in the central nervous system in AIDS patients. However, neurological abnormalities still remain important causes of mortality and morbidity in developing countries. In Brazil, cerebral toxoplasmosis is the most common cerebral mass lesion in AIDS patients. For these reasons, early, inexpensive, and sensitive diagnostic tests must be evaluated. The aim of this study was to evaluate PCR, using cerebrospinal fluid (CSF) samples to detect Toxoplasma gondii DNA, and to determine if the association of PCR with immunological assays can contribute to a timely diagnosis. We studied two sample groups. First, we analyzed stored CSF samples from 29 newborns and from 39 adults with AIDS without a definitive diagnosis of toxoplasmosis. The goal of this step was to standardize the methodology with a simple and economical procedure to recover the T. gondii DNA. Next, we prospectively evaluated CSF samples from 12 AIDS patients with a first episode of cerebral toxoplasmosis and 18 AIDS patients with other neurological opportunistic diseases and without previous cerebral toxoplasmosis. In all PCR samples, an indirect immunofluorescent assay and an enzyme-linked immunosorbent assay were performed. Samples from all patients with cerebral toxoplasmosis presented positive PCR results (sensitivity, 100%), and a sample from one of the 18 AIDS patients with other neurological diseases also presented positive PCR results (specificity, 94.4%). These findings suggest the clinical utility of PCR in the diagnosis of cerebral toxoplasmosis in developing countries.

Bordetella pertussis was diagnosed in a human immunodeficiency virus-infected patient by a newly developed method in which bacterial DNA is amplified directly from sputum Gram-stained slides. The validation of the method is described along with an additional new PCR-based assay that can distinguish between B. pertussis and Bordetella holmesii.

To compare the performance of the BACTEC 13A (Becton Dickinson, Sparks, Md.), BACTEC MYCO/F LYTIC (Becton Dickinson), BacT/ALERT MB (bioMérieux, Durham, N.C.), and ISOLATOR 10 lysis-centrifugation (Wampole Laboratories, Cranbury, N.J.) systems for detection of mycobacteremia in adults, we inoculated 5-ml aliquots of blood from patients with suspected mycobacteremia into the bottle or tube required for each system. Of 600 sets tested, 85 (14%) yielded Mycobacterium avium complex (MAC) and 9 (2%) yielded other species of mycobacteria. Of 26 complete (three bottles and one tube) adequately filled (5 +/- 1 ml) sets from which MAC was recovered, BACTEC 13A was positive for 19 (73%), BACTEC MYCO/F LYTIC was positive for 21 (81%), BacT/ALERT MB was positive for 22 (85%), and ISOLATOR 10 was positive for 21 (81%). Of the six possible two-way comparisons, the mean times to detection for the recovery of MAC from each bottle in positive adequately paired sets were 15.3 days for BACTEC 13A versus 12.8 days for MYCO/F LYTIC for 33 of 340 pairs, 14.1 days for BACTEC 13A versus 11.6 days for BacT/ALERT MB for 38 of 380 pairs, 12.6 days for BACTEC 13A versus 20.0 days for ISOLATOR 10 for 26 of 261 pairs, 12.8 days for BACTEC MYCO/F LYTIC versus 11.0 days for BacT/ALERT MB for 33 of 340 pairs, 13.2 days for BACTEC MYCO/F LYTIC versus 20.4 days for ISOLATOR 10 for 24 of 230 pairs, and 9.9 days for BacT/ALERT MB versus 19.0 days for ISOLATOR 10 for 24 of 257 pairs. There were no significant differences in yields between the systems. However, the mean time to detection differed significantly among the systems. The time to detection was shortest for BacT/ALERT MB, followed by BACTEC MYCO/F LYTIC and BACTEC 13A and then ISOLATOR 10. Although the numbers were too small for statistical comparison, the time to detection was substantially shorter for MAC than for Mycobacterium tuberculosis complex in the liquid systems. The continuously monitored systems (BACTEC MYCO/F LYTIC and BacT/ALERT MB) were as sensitive and, on balance, faster for the detection of MAC bacteremia than were the heretofore standard manual ISOLATOR 10 and radiometric BACTEC 13A systems.

A new real-time PCR assay for quantitation of Encephalitozoon intestinalis DNA was developed which used a TaqMan fluorescent probe for specific detection. Serial dilutions of E. intestinalis spore suspensions obtained from tissue culture were used as external standards. The detection limit of the technique was 20 spores per ml, with a good interassay reproducibility (coefficient of variation of 7.1% for the suspension containing 20 spores/ml, 5.0% for the suspension containing 75 spores/ml and below 3.5% for higher concentrations). Quantitative detection of E. intestinalis DNA was similar whether the serial dilutions of spores were made in distilled water or in a stool suspension, allowing the use of the assay for stool specimens. The assay was then applied to 14 clinical specimens from 8 immunocompromised patients with proven E. intestinalis infection. The quantitation of the parasitic burden was achieved in stools, blood, urine, tissue biopsies, and bronchopulmonary specimens. The highest parasitic burdens were noted in stools, urine, and bronchopulmonary specimens, reaching 10(5) to 10(6) spores/g or ml. Dissemination of the infection was also evidenced in some patients by demonstration of E. intestinalis DNA in blood and serum. We conclude that real-time PCR is a valuable tool for quantitation of E. intestinalis burden in clinical specimens.

Toxoplasma gondii is a common life-threatening opportunistic infection. We used experimental murine T. gondii infection to optimize the PCR for diagnostic use, define its sensitivity, and characterize the time course and tissue distribution of experimental toxoplasmosis. PCR conditions were adjusted until the assay reliably detected quantities of DNA derived from less than a single parasite. Forty-two mice were inoculated intraperitoneally with T. gondii tachyzoites and sacrificed from 6 to 72 h later. Examination of tissues with PCR and histology revealed progression of infection from blood to lung, heart, liver, and brain, with PCR consistently detecting parasites earlier than microscopy and with no false-positive results. We then evaluated the diagnostic value of this PCR assay in human patients. We studied cerebrospinal fluid and serum samples from 12 patients with AIDS and confirmed toxoplasmic encephalitis (defined as positive mouse inoculation and/or all of the Centers for Disease Control clinical diagnostic criteria), 12 human immunodeficiency virus-infected patients with suspected cerebral toxoplasmosis who had neither CDC diagnostic criteria nor positive mouse inoculation, 26 human immunodeficiency virus-infected patients with other opportunistic infections and no signs of cerebral toxoplasmosis, and 18 immunocompetent patients with neurocysticercosis. Eleven of the 12 patients with confirmed toxoplasmosis had positive PCR results in either blood or cerebrospinal fluid samples (6 of 9 blood samples and 8 of 12 cerebrospinal fluid samples). All samples from control patients were negative. This study demonstrates the high sensitivity, specificity, and clinical utility of PCR in the diagnosis of toxoplasmic encephalitis in a resource-poor setting.

Penicilliosis, caused by the dimorphic fungus Penicillium marneffei, is an important opportunistic systemic fungal infection affecting immunocompromised individuals living in areas where penicilliosis is endemic. We have demonstrated previously that a urinary enzyme-linked immunosorbent assay (ELISA) with purified rabbit polyclonal antibody against killed whole-fission-form arthroconidia of P. marneffei was specific and highly sensitive for the diagnosis of penicilliosis. In this study, a dot blot ELISA and a latex agglutination (LA) test were developed with the same polyclonal antibody and compared with the ELISA for the detection of P. marneffei urinary antigen. Urine specimens from 37 patients with culture-proven penicilliosis and 300 controls (52 healthy subjects and 248 hospitalized patients without penicilliosis) were tested. Antigen was detected in urine from all 37 (100%) penicilliosis patients by the LA test, 35 (94.6%) penicilliosis patients by the dot blot ELISA, and 36 (97.3%) penicilliosis patients by the ELISA. False-positive results were found by the three assays for 2 (0.7%), 8 (2.7%), and 6 (2%) of 300 controls, respectively. The overall sensitivities of the diagnostic tests were as follows: dot blot ELISA, 94.6%; ELISA, 97.3%; and LA test, 100% (specificities, 97.3, 98, and 99.3%, respectively). The LA test is simple, robust, rapid, and convenient and should prove to be an important addition to the existing diagnostic tests for penicilliosis.

We compared the clinical and laboratory features of human immunodeficiency virus (HIV)- and non-HIV-infected patients with penicilliosis marneffei. HIV-infected patients had a higher incidence of fungemia. A total of 85.7% of the HIV-negative patients had underlying diseases including hematologic malignancies or had received therapy with corticosteroids or cytotoxic agents. By a Penicillium marneffei-specific mannoprotein Mp1p enzyme-linked immunosorbent assay, serum antigen titers were found to be higher in HIV-positive patients, whereas serum antibody levels were found to be higher in HIV-negative patients.

We report the first case of Mycobacterium lentiflavum disseminated infection in a human immunodeficiency virus-infected patient. Conventional identification procedures failed to identify the mycobacterial strain, but sequencing of the 16S rRNA gene led to the species identification. Furthermore, we describe here the analysis of the 16S-23S rRNA internal transcribed spacer sequence of M. lentiflavum.

A quality assurance program was established by the Pediatric Pulmonary and Cardiovascular Complications of Vertically Transmitted Human Immunodeficiency Virus Type 1 Infection Study Group for monitoring cytomegalovirus (CMV) antibody and culture results obtained from nine different participating laboratories. Over a 3-year period, every 6 months, each laboratory was sent by the designated reference laboratory six coded samples: three urine samples for CMV detection and three serum samples for CMV immunoglobulin G (IgG) and IgM antibody determination. Overall, the participating laboratories exhibited the following composite performance statistics, relative to the reference laboratory (sensitivity and specificity, respectively): 100 and 97.4% for CMV cultures, 95.5 and 94.4% for CMV IgG antibody assays, and 92.6 and 90.2% for CMV IgM assays. The practice of having individual laboratories use different commercial methods and reagents for CMV detection and antibody determination was successfully monitored and provided useful information on the comparable performance of different assays.

We report on a case of mixed infection caused by two species of Fusarium in a human immunodeficiency virus-positive patient with lymphoma who was neutropenic due to chemotherapy. The patient showed the typical signs of a disseminated fusarial infection, with Fusarium solani isolated from skin lesions and F. verticillioides isolated from blood. The report discusses how difficult it is to make an accurate diagnosis when an immunosuppressed patient is infected with more than one fungal species, especially when the species are morphologically very similar.

We used a multiplex nested-PCR assay for the simultaneous detection in cerebrospinal fluid (CSF) of five human herpesviruses (HVs) (cytomegalovirus [CMV], Epstein-Barr virus [EBV], varicella-zoster virus [VZV], herpes simplex virus [HSV], and human herpesvirus 6 [HHV-6]) in a clinical evaluation of human immunodeficiency virus (HIV)-infected patients with neurological disorders. This method, which has the advantages of being rapid and economical, would be of particular interest for the diagnosis of neurological syndromes caused by more than one HV. We studied 251 CSF samples from 219 patients. HV DNA was demonstrated in 93 (37%) of the CSF samples (34% of the patients). CMV was the HV most frequently detected in our patients (25%), while EBV, VZV, HSV, and HHV-6 DNAs were present in significantly fewer cases (7, 4, 3, and 1%, respectively). When results were compared with the final etiological diagnoses of the patients, the multiplex HV PCR showed high specificity for the diagnosis of CMV and VZV neurological diseases and for cerebral lymphoma (0.95, 0.97, and 0.99, respectively). The sensitivity of the assay was high for CMV disease (0.87), was low for cerebral lymphoma (0.33), and was not evaluable for VZV disease due to the small number of patients with this diagnosis. Nevertheless, detection of VZV DNA had possible diagnostic value in four of the nine cases, and EBV DNA amplification always predicted the diagnosis of cerebral lymphoma in patients with cerebral masses. Detection of HSV DNA was frequently associated with CMV amplification and fatal encephalitis. HHV-6 was not considered to have a pathogenetic role in the three cases in which it was detected. This multiplex HV PCR assay is a specific and clinically useful method for the evaluation of HIV-infected patients with neurological disorders related to HV.

A nonchromogenic Mycobacterium species was isolated from an AIDS patient with acute lymphadenitis. On the basis of the results of conventional tests, the strain appeared to be an atypical nonphotochromogenic Mycobacterium kansasii strain. Sequencing of the 16S rRNA gene revealed a unique nucleic acid sequence, suggesting that the isolate represents an undescribed pathogenic species.

A total of 513 blood specimens, predominantly from organ transplant recipients, human immunodeficiency virus-positive patients, and bone marrow transplant recipients, were tested for cytomegalovirus (CMV) by culture and pp65 antigenemia across four test sites. Peripheral blood leukocytes were examined by using both the Biotest CMV Brite and the Bartels/Argene CMV Antigenemia kits. A total of 109 specimens were positive for CMV, 106 (97%) were positive by antigenemia, and 34 (31%) were positive by culture. According to the manufacturers' instructions, 150,000 cells were applied per slide for the Biotest kit and 200,000 cells per slide for the Bartels kit. A total of 93 specimens (88%) were positive by the Biotest kit, and 86 (81%) were positive by the Bartels kit. In specimens found to be positive by only one kit, the positive cell counts were low (median, 1; range, 1 to 7). When the data from all four sites were combined and analyzed, there was no statistical difference between the performance of the two kits; the Biotest and Bartels kits were found to be equivalent in sensitivity, specificity, and positive and negative predictive values for the detection of CMV pp65 antigenemia.

The genotypes and susceptibilities to fluconazole of 78 strains of the human pathogenic yeast Candida albicans were compared. The strains comprised two sets of samples from Durham, N.C.: one from patients infected with the human immunodeficiency virus (HIV) and the other from healthy volunteers. For each strain, the MIC of fluconazole was determined by the standard National Committee for Clinical Laboratory Standards protocol. Genotypes were determined by PCR fingerprinting with five separate primers. The analysis revealed little evidence for genotypic clustering according to HIV status or body site. However, a small group of fluconazole-resistant strains isolated from patients infected with HIV formed a distinct cluster. In addition, two fluconazole-resistant strains were isolated from individuals who never took fluconazole, one from a patient infected with HIV and the other from a healthy person. The results suggest both clonal and spontaneous origins of fluconazole resistance in C. albicans.

A total of 22 Cryptosporidium isolates from human immunodeficiency virus-infected patients from Kenya, Switzerland, and the United States were examined at three genetic loci: the 18S ribosomal DNA, HSP-70, and acetyl coenzyme A synthetase genes. Four distinct Cryptosporidium genotypes were identified: (i) the Cryptosporidium parvum "human" genotype, (ii) the C. parvum "cattle" genotype, (iii) Cryptosporidium felis, and (iv) Cryptosporidium meleagridis. This is the first report of C. meleagridis in a human host. These results and those of others indicate that immunocompromised individuals are susceptible to a wide range of Cryptosporidium species and genotypes. Future studies are required to understand the full public health significance of Cryptosporidium genotypes and species in immunocompromised hosts.

The purpose of this study was to determine the sensitivity and specificity of three different methods of cytomegalovirus (CMV) detection for AIDS patients at risk for CMV retinitis. Patients with CD4(+) counts of <100/microl and negative baseline screening eye examinations were tested for CMV infection by (i) pp65 antigenemia expression in leukocytes, (ii) the Digene Hybrid Capture CMV DNA System, and (iii) the Roche Amplicor Qualitative PCR Test. The incidence of CMV retinitis in our study of 296 patients at the Medical Center of Louisiana-New Orleans HIV Outpatient Clinic was 7. 2 per 100 person-years (a total of 20 episodes in 18 patients from April 1997 to February 1999). Receiver operating characteristic curves were calculated for each assay to determine optimal cutoff points which maximized the sensitivity and specificity of each assay. The sensitivities of the assays compared to the eye examinations were 80% for the pp65 antigenemia assay (cutoff, >0 cell per 1.5 x 10(5) leukocytes), 85% for the Digene assay (cutoff, 1,400 genome copies/ml of whole blood), and 60% for the Amplicor assay. The specificities of the assays were 84, 84, and 87%, respectively. The Digene assay with a cutoff of >/=1,400 genome copies/ml gave optimal sensitivity and specificity and was found to have predictive values equal to those of the more technically cumbersome antigenemia assay.

In the present prospective study, five blood tests for detection of human cytomegalovirus (HCMV), nucleic acid sequence-based amplification (NASBA) for detection of early (immediate-early antigen) and late (pp67) mRNA, PCR for detection of HCMV DNA (DNA PCR), culture, and pp65 antigenemia assay, and culture and DNA PCR of urine and throat swab specimens were compared for their abilities to predict the development of disease caused by HCMV (HCMV disease). Of 101 human immunodeficiency virus (HIV)-infected patients with

Several hybridomas producing antibodies detected by indirect immunofluorescence antibody test (IFAT) were established by fusion of mouse myeloma SP2/O with spleen cells from BALB/c mice immunized against whole spores (protocol 1) or chitinase-treated spores (protocol 2) of Enterocytozoon bieneusi and were cloned twice by limiting dilutions. Two monoclonal antibodies (MAbs), 3B82H2 from protocol 1, isotyped as immunoglobulin M (IgM), and 6E52D9 from protocol 2, isotyped as IgG, were expanded in both ascites and culture. IFAT with the MAbs showed that both MAbs reacted exclusively with the walls of the spores of E. bieneusi, strongly staining the surface of mature spores, and produced titers of greater than 4,096. Immunogold electron microscopy confirmed the specific reactivities of both antibodies. No cross-reaction, either with the spores of the other intestinal microsporidium species Encephalitozoon intestinalis or with yeast cells, bacteria, or any other intestinal parasites, was observed. The MAbs were used to identify E. bieneusi spores in fecal specimens from patients suspected of having intestinal microsporidiosis. The IFAT was validated against standard staining methods (Chromotrope 2R and Uvitex 2B) and PCR. We report here the first description and characterization of two MAbs specific for the spore wall of E. bieneusi. These MAbs have great potential for the demonstration and species determination of E. bieneusi, and their application in immunofluorescence identification of E. bieneusi in stool samples could offer a new diagnostic tool for clinical laboratories.

Cobas Amplicor CMV Monitor (CMM) and Quantiplex CMV bDNA 2.0 (CMV bDNA 2.0), two new standardized and quantitative assays for the detection of cytomegalovirus (CMV) DNA in plasma and peripheral blood leukocytes (PBLs), respectively, were compared to the CMV viremia assay, pp65 antigenemia assay, and the Amplicor CMV test (P-AMP). The CMV loads were measured in 384 samples from 58 human immunodeficiency virus (HIV) type 1-infected, CMV-seropositive subjects, including 13 with symptomatic CMV disease. The assays were highly concordant (agreement, 0.88 to 0.97) except when the CMV load was low. Quantitative results for plasma and PBLs were significantly correlated (Spearman rho = 0.92). For PBLs, positive results were obtained 125 days before symptomatic CMV disease by CMV bDNA 2.0 and 124 days by pp65 antigenemia assay, whereas they were obtained 46 days before symptomatic CMV disease by CMM and P-AMP. At the time of CMV disease diagnosis, the sensitivity, specificity, and positive and negative predictive values of CMV bDNA 2.0 were 92.3, 97.8, 92.3, and 97.8%, respectively, whereas they were 92.3, 93.3, 80, and 97. 8%, respectively, for the pp65 antigenemia assay; 84.6, 100, 100, and 95.7%, respectively, for CMM; and 76.9, 100, 100, and 93.8%, respectively, for P-AMP. Considering the entire follow-up, the sensitivity, specificity, and positive and negative predictive values of CMV bDNA 2.0 were 92.3, 73.3, 52.1, and 97.1%, respectively, whereas they were 100, 55.5, 39.4, and 100%, respectively, for the pp65 antigenemia assay; 92.3, 86.7, 66.7, and 97.5%, respectively, for CMM; and 84.6, 91.1, 73.3, and 95.3%, respectively, for P-AMP. Detection of CMV in plasma is technically easy and, despite its later positivity (i.e., later than in PBLs), can provide enough information sufficiently early so that HIV-infected patients can be effectively treated. In addition, these standardized quantitative assays accurately monitor the efficacy of anti-CMV treatment.

Of the several microsporidia that infect humans, Enterocytozoon bieneusi is known to cause a gastrointestinal disease whereas Encephalitozoon intestinalis causes both a disseminated and an intestinal disease. Although several different staining techniques, including the chromotrope technique and its modifications, Uvitex 2B, and the quick-hot Gram-chromotrope procedure, detect microsporidian spores in fecal smears and other clinical samples, they do not identify the species of microsporidia. A need for an easily performed test therefore exists. We reevaluated 120 stool samples that had been found positive for microsporidia previously, using the quick-hot Gram-chromotrope technique, and segregated them into two groups on the basis of spore size. We also screened the smears by immunofluorescence microscopy, using a polyclonal rabbit anti-E. intestinalis serum at a dilution of 1:400. Spores in 29 (24.1%) of the 120 samples fluoresced brightly, indicating that they were E. intestinalis spores. No intense background or cross-reactivity with bacteria, yeasts, or other structures in the stool samples was seen. Additionally, the numbers of spores that fluoresced in seven of these samples were substantially smaller than the numbers of spores that were present in the stained smears, indicating that these samples were probably derived from patients with mixed infections of Enterocytozoon bieneusi and E. intestinalis. Because a 1:400 dilution of this serum does not react with culture-grown Encephalitozoon hellem, Encephalitozoon cuniculi, or Vittaforma corneae or with Enterocytozoon bieneusi spores in feces, we concluded that an immunofluorescence test using this serum is a good alternative for the specific identification of E. intestinalis infections.

The roles of Mycoplasma genitalium and Ureaplasma urealyticum in nongonococcal urethritis are not yet well established. The aim of this study was to determine the presence of these microorganisms in the urethral tracts of 187 human immunodeficiency virus type 1 (HIV-1)-infected male patients with no clinical signs of urethritis. The results indicate that the prevalence of M. genitalium and U. urealyticum was higher in AIDS patients than in asymptomatic, HIV-1-infected patients and in healthy individuals. The high rate of mycoplasmas and ureaplasmas detected in AIDS patients, in the absence of urethritis, argues against major roles in causing disease at the urethral mucosal level for these microorganisms.

JC polyomavirus (JCV) is the causative agent of progressive multifocal leukoencephalopathy (PML), a central nervous system infection that mainly affects AIDS patients. The extensive application of highly active antiretroviral therapy (HAART) is leading to the appearance of "long-term" survival PML patients. A reliable and feasible qualitative-quantitative test for both the detection of JCV and follow-up of its viral burden in this emerging group of patients is clearly required. With this aim, a dual qualitative-quantitative nested PCR is presented in this study for the analysis of JCV DNA in cerebrospinal fluid (CSF). Two newly designed internal controls, one competitive and the other noncompetitive, have been constructed to adapt this PCR to either measure the JCV burden or to allow a highly confident determination of JCV presence or clearance. The analytical sensitivity of the technique allows the detection of 0.01 fg (three genomes) of JCV DNA. Its qualitative application has been evaluated by analyzing single CSF samples from a group of 17 patients with PML and a control group of 20 patients with diverse neurological conditions other than PML, yielding sensitivity and specificity values of 100 and 90%, respectively. The quantitative application has been evaluated in vitro in blind tests with samples including serial dilutions of JCV, and in all cases the samples were successfully ordered considering the JCV titer. The dual quantitative-qualitative application offered by this nested PCR may provide an answer to the new requirements for evaluating and finely monitoring PML in AIDS patients receiving HAART.

Histoplasmosis is an important systemic fungal infection, particularly among immunocompromised individuals, who may develop a progressive disseminated form which is often fatal if it is untreated. In such patients, the detection of antibody responses for both diagnosis and follow-up may be of limited use, whereas the detection of Histoplasma capsulatum var. capsulatum antigens may provide a more practical approach. We have recently described an inhibition enzyme-linked immunosorbent assay (ELISA) for the detection in patients' sera of a 69- to 70-kDa H. capsulatum var. capsulatum-specific antigen which appears to be useful in diagnosis. To investigate its potential for the follow-up of histoplasmosis patients during treatment, antigen titers in the sera of 16 patients presenting with different clinical forms of histoplasmosis were monitored at regular intervals for up to 80 weeks. Sera from four of five patients with the acute form of the disease showed rapid falls in antigenemia, becoming antigen negative by week 14 (range, weeks 10 to 16). Sera from four patients with disseminated histoplasmosis showed falls in antigen levels; three of them became antigen negative by week 32; the fourth patient became negative by week 48. In contrast, antigen titers in four of six AIDS patients with the disseminated form of the disease remained positive throughout follow-up. Sera from only one patient who presented with the chronic form of the disease were analyzed, and this individual's serum became antigen negative by week 9. The inhibition ELISA is shown to be of particular use in the monitoring of non-AIDS patients with the acute and disseminated forms of the disease and may complement existing means of follow-up.

One hundred eighteen Candida clinical isolates from human immunodeficiency virus-infected patients were tested for their susceptibilities to fluconazole and itraconazole by Fungitest and the National Committee for Clinical Laboratory Standards MIC method. Fungitest results depended on both yeast species and antifungal agents. This test is able to detect sensitive strains (97% agreement with results of the MIC method in tests with fluconazole and 84% agreement in tests with itraconazole) but has a poor capacity to detect resistant strains (26% agreement in tests with fluconazole and 5% agreement in tests with itraconazole).