Detection of Mycobacterium tuberculosis in Sputum by Gas Chromatography-Mass Spectrometry of Methyl Mycocerosates Released by Thermochemolysis

Tuberculosis requires rapid diagnosis to prevent further transmission and allow prompt administration of treatment. Current methods for diagnosing pulmonary tuberculosis lack sensitivity are expensive or are extremely slow. The identification of lipids using gas chromatography- electron impact mass spectrometry (GC-EI/MS) could provide an alternative solution. We have studied mycocerosic acid components of the phthiocerol dimycocerosate (PDIM) family of lipids using thermochemolysis GC-EI/MS. To facilitate use of the technology in a routine diagnostic laboratory a simple extraction procedure was employed where PDIMs were extracted from sputum using petroleum ether, a solvent of low polarity. We also investigated a method using methanolic tetramethylammonium hydroxide, which facilitates direct transesterification of acidic components to methyl esters in the inlet of the GC-MS system. This eliminates conventional chemical manipulations allowing rapid and convenient analysis of samples. When applied to an initial set of 40 sputum samples, interpretable results were obtained for 35 samples with a sensitivity relative to culture of 94% (95%CI: 69.2,100) and a specificity of 100% (95%CI: 78.1,100). However, blinded testing of a larger set of 395 sputum samples found the assay to have a sensitivity of 61.3% (95%CI: 54.9,67.3) and a specificity of 70.6% (95%CI: 62.3,77.8) when compared to culture. Using the results obtained we developed an improved set of classification criteria, which when applied in a blinded re-analysis increased the sensitivity and specificity of the assay to 64.9% (95%CI: 58.6,70.8) and 76.2% (95%CI: 68.2,82.8) respectively. Highly variable levels of background signal were observed from individual sputum samples that inhibited interpretation of the data. The diagnostic potential of using thermochemolytic GC-EI/MS of PDIM biomarkers for diagnosis of tuberculosis in sputum has been established; however, further refinements in sample processing are required to enhance the sensitivity and robustness of the test

An Autopsy Study Describing Causes of Death and Comparing Clinico-Pathological Findings among Hospitalized Patients in Kampala, Uganda

Background: Information on causes of death in HIV-infected patients in Sub-Saharan Africa is mainly derived from observational cohort and verbal autopsy studies. Autopsy is the gold standard to ascertain cause of death. We conducted an autopsy study to describe and compare the clinical and autopsy causes of death and contributory findings in hospitalized HIV-infected and HIV-uninfected patients in Uganda. Methods: Between May and September 2009 a complete autopsy was performed on patients that died on a combined infectious diseases gastroenterology ward in Mulago Hospital in Kampala, Uganda. Autopsy cause of death and contributing findings were based on the macro- and microscopic post-mortem findings combined with clinical information. Clinical diagnoses were reported by the ward doctor and classified as confirmed, highly suspected, considered or not considered, based on information derived from the medical chart. Results are reported according to HIV serostatus. Results: Fifty-three complete autopsies were performed in 66 % HIV-positive, 21 % HIV-negative and 13 % patients with an unknown HIV serological status. Infectious diseases caused death in 83 % of HIV-positive patients, with disseminated TB as the main diagnosis causing 37 % of deaths. The spectrum of illness and causes of death were substantially different betwee

Lipoarabinomannan in urine during tuberculosis treatment: association with host and pathogen factors and mycobacteriuria

BACKGROUND: Detection of lipoarabinomannan (LAM), a Mycobacterium tuberculosis (Mtb) cell wall antigen, is a potentially attractive diagnostic. However, the LAM-ELISA assay has demonstrated variable sensitivity in diagnosing TB in diverse clinical populations. We therefore explored pathogen and host factors potentially impacting LAM detection. METHODS: LAM-ELISA assay testing, sputum smear and culture status, HIV status, CD4 cell count, proteinuria and TB outcomes were prospectively determined in adults diagnosed with TB and commencing TB treatment at a South African township TB clinic. Sputum TB isolates were characterised by IS61110-based restriction fragment length polymorphism (RFLP) and urines were tested for mycobacteriuria by Xpert® MTB/RIF assay. RESULTS: 32/199 (16.1%) of patients tested LAM-ELISA positive. Median optical density and proportion testing LAM positive remained unchanged during 2 weeks of treatment and then declined over 24 weeks. LAM was associated with positive sputum smear and culture status, HIV infection and low CD4 cell counts but not proteinuria, RFLP strain or TB treatment outcome. The sensitivity of LAM for TB in HIV-infected patients with CD4 counts of ≥ 200, 100-199, 50-99, and < 50 cells/μl, was 15.2%, 32%, 42.9%, and 69.2% respectively. Mycobacteriuria was found in 15/32 (46.9%) of LAM positive patients and in none of the LAM negative controls. CONCLUSIONS: Urinary LAM was related to host immune factors, was unrelated to Mtb strain and declined steadily after an initial 2 weeks of TB treatment. The strong association of urine LAM with mycobacteriuria is a new finding, indicating frequent TB involvement of the renal tract in advanced HIV infection

Comparison of a Clinical Prediction Rule and a LAM Antigen-Detection Assay for the Rapid Diagnosis of TBM in a High HIV Prevalence Setting

Background/Objective: The diagnosis of tuberculous meningitis (TBM) in resource poor TB endemic environments is challenging. The accuracy of current tools for the rapid diagnosis of TBM is suboptimal. We sought to develop a clinical-prediction rule for the diagnosis of TBM in a high HIV prevalence setting, and to compare performance outcomes to conventional diagnostic modalities and a novel lipoarabinomannan (LAM) antigen detection test (Clearview-TB (R)) using cerebrospinal fluid (CSF).Methods: Patients with suspected TBM were classified as definite-TBM(CSF culture or PCR positive), probable-TBM and non-TBM.Results: Of the 150 patients, 84% were HIV-infected (median [IQR] CD4 count = 132 [54; 241] cells/mu l). There were 39, 55 and 54 patients in the definite, probable and non-TBM groups, respectively. The LAM sensitivity and specificity (95% CI) was 31% (17; 48) and 94% (85; 99), respectively (cut-point >= 0.18). By contrast, smear-microscopy was 100% specific but detected none of the definite-TBM cases. LAM positivity was associated with HIV co-infection and low CD4 T cell count (CD4200 cells/mu l; p = 0.03). The sensitivity and specificity in those with a CD4= 6 derived from multivariate analysis had a sensitivity and specificity (95% CI) of 47% (31; 64) and 98% (90; 100), respectively. When LAM was combined with the clinical-prediction-rule, the sensitivity increased significantly (p<0.001) to 63% (47; 68) and specificity remained high at 93% (82; 98).Conclusions: Despite its modest sensitivity the LAM ELISA is an accurate rapid rule-in test for TBM that has incremental value over smear-microscopy. The rule-in value of LAM can be further increased by combination with a clinical-prediction rule, thus enhancing the rapid diagnosis of TBM in HIV-infected persons with advanced immunosuppression

Cancer Screening and Cancer Mortality in Nevis, West Indies

Objective: Cancer screening is one approach that can reduce morbidity and mortality through early detection of pre-cancers; however anxiety, fear, and lack of information are important barriers to universal cancer screening in the Caribbean. Nevis is a small island located in the Eastern Caribbean. We report available data on screening prevalence and cancer mortality for four common cancers: cervical, breast, colon and prostate. Methods: Demographic information, screening utilization (when available) and cancer mortality statistics were obtained from the Charlestown Health Clinic and the annual reports from the Ministry of Health. Moreover, physicians and key stakeholders were interviewed to assess current guidelines for cancer screening as well as to indicate areas of need. Results: Cervical cancer screening is under-utilized in Nevis. Between 2001 and 2007, the overall prevalence of Pap testing fluctuated minutely (mean: 6.87%, range: 6.06 – 7.41%). Systematic screening for breast, colon, and prostate cancer is not routinely performed, thus utilization rates are not available. Cancer mortality varied slightly between 2002 and 2006; prostate cancer had the highest overall crude mortality rate (30.6 deaths/100 000 persons). Physician interviews revealed that adherence to US and European cancer screening guidelines are inappropriate for their population of patients. Breast and prostate cancers are frequently diagnosed in these geographic areas before the age when cancer screening is currently recommended. Conclusion: Cancer is perceived as an important health problem by physicians, key stakeholders and citizens, however cancer screening is under-utilized in Nevis. Future research should focus on generating screening guidelines that are relevant for this population, as well as methods to promote screening. "Pesquisaje de Cáncer y Mortalidad por Cáncer en Nevis, West Indies" RESUMEN Objetivo: El pesquisaje de cáncer es un método que puede reducir la morbilidad y la mortalidad mediante la detección precoz de pre-cánceres. Sin embargo, la ansiedad, el miedo, y la falta de información son barreras importantes que se erigen frente al pesquisaje universal del cáncer en el Caribe. Nevis es una pequeña isla situada en el Caribe Oriental. Reportamos datos disponibles sobre la prevalencia del pesquisaje y la mortalidad por cáncer en relación con cuatro tipos de cánceres comunes: el cervical, y el de mamas, colon y próstata. Métodos: La información demográfica, la utilización de pesquisaje (de hallarse disponible) y las estadísticas sobre la mortalidad por cáncer, se obtuvieron de la Clínica de la Salud de Charlestown, y los reportes anuales del Ministerio de Salud. Además, los médicos y las partes interesadas claves, fueron entrevistados a fin de evaluar las guías actuales para el pesquisaje del cáncer así como para indicar las áreas de necesidad. Resultados: El pesquisaje del cáncer cervical es subutilizado en Nevis. Entre 2001 y 2007, la prevalencia general de la prueba citológica tuvo una fluctuación mínima (promedio: 6.87%, rango: 6.06 – 7.41%). El pesquisaje sistemático del cáncer de mamas, colon, y próstata no se realiza como rutina, y por consiguiente no existen tasas de utilización disponibles. La mortalidad por cáncer varió ligeramente entre 2002 y 2006. El cáncer de próstata tuvo la tasa de mortalidad cruda general más alta (30.6 muertes/100 000 personas). Las entrevistas médicas revelaron que las guías de pesquisaje en USA y Europa son inapropiadas para la población de pacientes. Los cánceres de mamas y próstata son diagnosticados con frecuencia en estas áreas geográficas antes de la edad en que se recomienda el pesquisaje actualmente. Conclusión: El cáncer es percibido como un importante problema de salud por los médicos, las partes interesadas claves, y los ciudadanos. No obstante, el pesquisaje del cáncer es subutilizado en Nevis. Las investigaciones futuras deben centrarse en producir guías de pesquisaje que sean pertinentes para esta población, así como en métodos de promover el pesquisaje

High Frequency of Syncytium-Inducing and CXCR4-Tropic Viruses among Human Immunodeficiency Virus Type 1 Subtype C-Infected Patients Receiving Antiretroviral Treatment

Human immunodeficiency virus type 1 (HIV-1) subtype C viruses have been found to almost exclusively use the chemokine receptor CCR5 as a coreceptor for entry, even in patients with advanced AIDS. We have characterized subtype C virus isolates from 28 patients from Harare, Zimbabwe, 20 of whom were receiving antiretroviral treatment. Virus from 10 of the treated patients induced syncytium formation (SI virus) when cultured with MT2 cells. Only non-syncytium-inducing (NSI) virus was cultured from the peripheral blood mononuclear cells of the eight patients who had not received treatment. The majority of these subtype C SI viruses were capable of using both CCR5 and CXCR4 as coreceptors for viral entry, and the consensus V3 loop sequences from the SI viruses displayed a high net charge compared to those of NSI viruses. While those on treatment had reverse transcriptase (RT) and protease mutations, there was no clear association between RT and protease drug resistance mutations and coreceptor tropism. These results suggest that CXCR4-tropic viruses are present within the quasispecies of patients infected with subtype C virus and that antiretroviral treatment may create an environment for the emergence of CXCR4 tropism

Microscopic-observation drug-susceptibility assay for the diagnosis of drug-resistant tuberculosis in Harare, Zimbabwe.

Limited data exist on use of the microscopic-observation drug-susceptibility (MODS) assay among persons suspected of MDR-TB living in high HIV-prevalence settings.We retrospectively reviewed available clinical and drug susceptibility data for drug-resistant TB suspects referred for culture and drug-susceptibility testing between April 1, 2011 and March 1, 2012. The diagnostic accuracy of MODS was estimated against a reference standard including Löwenstein-Jensen (LJ) media and manual liquid (BACTEC MGIT) culture. The accuracy of MODS drug-susceptibility testing (DST) was assessed against a reference standard absolute concentration method.One hundred thirty-eight sputum samples were collected from 99 drug-resistant TB suspects; in addition, six previously cultured MDR isolates were included for assessment of DST accuracy. Among persons with known HIV infection status, 39/59 (66%) were HIV-infected. Eighty-six percent of patients had a history of prior TB treatment, and 80% of individuals were on antituberculous treatment at the time of sample collection. M. tuberculosis was identified by reference standard culture among 34/98 (35%) MDR-TB suspects. Overall MODS sensitivity for M. tuberculosis detection was 85% (95% CI, 69-95%) and specificity was 93% (95% CI, 84-98%); diagnostic accuracy did not significantly differ by HIV infection status. Median time to positivity was significantly shorter for MODS (7 days; IQR 7-15 days) than MGIT (12 days; IQR 6-16 days) or LJ (28 days; IQR 21-35 days; p<0.001). Of 33 specimens with concurrent DST results, sensitivity of the MODS assay for detection of resistance to isoniazid, rifampin, and MDR-TB was 88% (95% CI, 68-97%), 96% (95% CI, 79-100%), and 91% (95% CI, 72-99%), respectively; specificity was 89% (95% CI, 52-100%), 89% (95% CI, 52-100%), and 90% (95% CI, 56-100%), respectively.In a high HIV-prevalence setting, MODS diagnosed TB and drug-resistant TB with high sensitivity and shorter turnaround time compared with standard culture and DST methods

Impact of Xpert MTB/RIF on Antiretroviral Therapy-Associated Tuberculosis and Mortality: A Pragmatic Randomized Controlled Trial.

GeneXpert® MTB/RIF (Xpert) is now widely distributed in high human immunodeficiency virus (HIV)/tuberculosis (TB)-burden countries. Yet, whether the test improves patient-important outcomes within HIV treatment programs in limited resource settings is unk