An investigation of fingerstick blood collection for point-of-care HIV-1 viral load monitoring in South Africa.

BACKGROUND: Viral load (VL) quantification is an important tool in determining newly developed drug resistance or problems with adherence to antiretroviral therapy (ART) in HIV-positive patients. VL monitoring is becoming the standard of care in many resource-limited settings. Testing in resource-limited settings may require sampling by fingerstick because of general shortages of skilled phlebotomists and the expense of venepuncture supplies and problems with their distribution. OBJECTIVE: To assess the feasibility and ease of collecting 150 µL capillary blood needed for the use of a novel collection device following a classic fingerstick puncture. METHODS: Patients were recruited by the study nurse upon arrival for routine ART monitoring at the Themba Lethu Clinic in Johannesburg, South Africa. Each step of the fingerstick and blood collection protocol was observed, and their completion or omission was recorded. RESULTS: One hundred and three patients consented to the study, of whom three were excluded owing to the presence of callouses. From a total of 100 patients who consented and were enrolled, 98% of collection attempts were successful and 86% of participants required only one fingerstick to successfully collect 150 µL capillary blood. Study nurse adherence to the fingerstick protocol revealed omissions in several steps that may lower the success rate of capillary blood collection and reduce the performance of a subsequent VL assay. CONCLUSION: The findings of this study support the feasibility of collecting 150 µL of capillary blood via fingerstick for point-of-care HIV-1 VL testing in a resource-limited setting.

High incidence of latent tuberculous infection among South African health workers: an urgent call for action.

SETTING: In South Africa, health care workers (HCWs) are at two-fold greater risk of acquiring tuberculosis (TB) disease than the general population. Few studies have evaluated the risk of incident tuberculous infection. OBJECTIVE: To determine the incidence and risk factors for latent tuberculous infection (LTBI) among HCWs and to compare the results of the interferon-gamma release assay (IGRA) with those of the tuberculin skin test (TST). DESIGN: HCWs, including medical students, underwent a TST and human immunodeficiency virus (HIV) and IGRA testing at baseline and 12 months, and IGRA at 6 months. The participants kept 12-month TB exposure logs. RESULTS: Among 199 participants (150 [76%] females, median age 31 years [range 20-61]), incident LTBI was documented using IGRA in 25/97 (26%; incident rate 29 cases/100 person-years [py], 95%CI 20-44) and using TST in 25/93 (27%; incident rate 29 cases/100 py, 95%CI 19-42). Agreement between TST and IGRA was poor (44.8%, κ = 0.23). Higher annual exposure to TB cases was reported among persons with LTBI than in those who were persistently IGRA-negative (81 cases, 95%CI 61-102 vs. 50 cases, 95%CI 43-57, P < 0.01). CONCLUSION: The high LTBI incidence and the association of incident LTBI with annual TB caseload among HCWs indicate that more effective TB infection control should be implemented in South African health care facilities.

A pilot evaluation of external quality assessment of GenoType MTBDRplus versions 1 and 2 using dried culture spot material.

Dried culture spots (DCS) of inactivated Mycobacteria strains designed as part of an external quality assessment (EQA) program for the GeneXpert system has applications to other molecular tuberculosis (TB) diagnostic platforms. DCS tested on the GenoType MTBDRplus and Mycobacterium CM assays performed well with MTBDRplus version 2 but require increased bacterial concentration for use with version 1.

Performance monitoring of mycobacterium tuberculosis dried culture spots for use with the GeneXpert system within a national program in South Africa.

The use of dried culture spots (DCSs) has been reported in the verification of GeneXpert instruments as being "fit for purpose" for the South African National implementation program. We investigated and compared the performance of the DCSs for verification across different bulk batches, testing the settings and cadre of staff, and the Xpert MTB/RIF assay version. Four bulk batches (V005 to V008) were used to prepare (i) 619 DCS panels for laboratory testing on G3 or G4 cartridges by a technologist, (ii) 13 DCS panels (batch V005) used for clinic verification on G3 cartridges by a nurse or lay counselor, and (iii) 20 DCS panels (batch V005) used for the verification of 10 GeneXpert 16 module instruments in mobile vehicles on the G3 cartridge performed by a scientist. The stabilities of the DCSs over 6 months at 4°C, room temperature, and 37°C were investigated. The mean cycle threshold (CT) and standard deviation (SD) for probe A were calculated. The proportions of variability in the CT values across bulk batches, assay versions, and settings and cadre of staff were determined using regression analysis. Overall, the DCSs demonstrated SDs of 3.3 (n = 660) for the G3 cartridges and 3.8 (n = 1,888) for the G4 cartridges, with an overall error rate of 1.5% and false rifampin resistance rate of 0.1%. The proportions of variability (R(2)) in the CT values explained by batch were 14%, by setting and cadre of staff, 5.6%, and by assay version, 4.2%. The most stable temperature in a period of up to 6 months was 37°C (SD, 2.7). The DCS is a robust product suitable for storage, transport, and use at room temperature for the verification of the GeneXpert instrument, and the testing can be performed by non-laboratory-trained personnel in nonlaboratory settings.

Performance of the Roche LightCycler real-time PCR assay for diagnosing extrapulmonary tuberculosis.

The Roche LightCycler mycobacterium detection molecular assay for Mycobacterium tuberculosis, M. avium, and M. kansasii, was applied to tissue specimens. It performed well on lymph node and cerebrospinal fluid specimens and less well on lung, liver, and bone marrow core biopsy specimens, but used in conjunction with a clinical suspicion of tuberculosis, it could augment patient management.

Dried culture spots for Xpert MTB/RIF external quality assessment: results of a phase 1 pilot study in South Africa.

Implementation of Xpert MTB/RIF requires quality assessment. A pilot program using dried culture spots (DCSs) of inactivated Mycobacterium tuberculosis is described. Of 274 DCS results received, 2.19% generated errors; the remainder yielded 100% correct Mycobacterium tuberculosis detection. The probe A cycle threshold (C(T)) variability of three DCS batches was ≤ 3.47. The study of longer-term DCS stability is ongoing.

Poor sensitivity of field rapid HIV testing: implications for mother-to-child transmission programme.

We validated rapid HIV tests among pregnant women in a clinical setting. Field testing was performed using First Response 1,2,3 or Standard Diagnostic and Pareekshak tests. Results were confirmed by third generation HIV ELISA. Discordant or negative, specimens were confirmed by RNA PCR and a fourth generation ELISA test. Sensitivity and specificity were 94.5% (CI: 85.8-98.2) and 100% for First Response; 87.5% (CI: 46.7-99.3) and 100% (CI: 87.7-100%) for Standard Diagnostic and 90.2% (CI: 81.2-95.4) and 100% (CI: 98-100%) for Pareekshak. These sensitivities were lower than laboratory validation which approached 100%. The low-field sensitivity results have implications for Prevention of Mother-to-Child Transmission services.

Evaluation of the performance of the automated NucliSENS easyMAG and EasyQ systems versus the Roche AmpliPrep-AMPLICOR combination for high-throughput monitoring of human immunodeficiency virus load.

This study presents the data of an evaluation of the automated Nuclisens easyMAG and EasyQ systems versus the Roche AmpliPrep-AMPLICOR combination for testing of high-volume human immunodeficiency virus (HIV) load. This represents a follow-up of a previous study investigating the performance of the real-time Nuclisens assay using the semiautomated NucliSENS miniMAG extraction procedure. Three hundred eighteen patient samples were analyzed using both methods. The easyMAG-EasyQ HIV type 1 system has a higher sensitivity and broader dynamic range than the Cobas AmpliPrep-AMPLICOR system when the standard Roche assay is used alone, 25 to 3,000,000 IU/ml versus 400 to 750,000 HIV RNA copies/ml, respectively. There was significant correlation between the assays (0.93; P < 0.0001), with good accuracy (percent similarity mean mu = 96%), good precision (percent similarity standard deviation = 4.97%), and overall good agreement with a low percent similarity coefficient of variation of 5.17 to 6.11%. Bland-Altman analysis revealed that the AMPLICOR assay generated higher values than the EasyQ combination, with 95% of results within clinically acceptable limits. The throughput of samples was greatly improved using the easyMAG-EasyQ system, allowing 144 samples to be processed within 6 h. The potential for contamination has been dramatically reduced using the automated extraction system. Additional negative controls have been added to the kit to monitor for contamination based on the South African experience. This assay thus presents a real option for monitoring HIV load assays in high-volume testing environments.

Evaluation of two commercially available, inexpensive alternative assays used for assessing viral load in a cohort of human immunodeficiency virus type 1 subtype C-infected patients from South Africa.

Although human immunodeficiency virus type 1 (HIV-1) RNA is the acknowledged "gold standard" marker for monitoring disease activity in patients receiving highly active antiretroviral therapy (HAART), it remains unaffordable in resource-constrained settings. The present study investigated two commercially available kits for the detection of HIV-1 viral load markers as more affordable alternatives to HIV-1 RNA quantitation. The greatly improved heat-denatured, signal-boosted HiSens HIV-1 p24 Ag Ultra kit (Perkin-Elmer) and the ExaVir Load Quantitative HIV-RT kit (Cavidi Tech AB) were compared with the Amplicor HIV-1 Monitor (version 1.5) assay (Roche Molecular Systems Inc.). A total of 117 samples containing HIV-1 subtype C were analyzed by all three methodologies. Eighty-nine of these samples represented serial measurements from 20 patients receiving HAART. The remaining samples analyzed were from a group of treatment-naive patients. The association between the p24 antigen assay and the RNA assay was fairly strong (R(2) = 0.686). The association between the reverse transcriptase (RT) quantitation assay and the RNA assay was strong (R(2) = 0.810). Both alternative assays seemed most useful for the serial monitoring of patients receiving HAART (n = 89 plasma samples from 20 patients), as all assays showed a statistically significant downward trend over time, with the trend being either linear or curvilinear. In addition, all three assays showed negative correlations with the CD4 count (CD4 count versus RNA load, r = -0.336 and P = 0.001; CD4 count versus p24 antigen level, r = -0.541 and P < 0.0001; CD4 count versus RT level, r = -0.358 and P = 0.0006). Still of major concern are both the lack of sensitivity and the wide degrees of variability of both assays. However, both assays provide a less expensive alternative to the Roche viral load assay and demonstrate the same trends during treatment.

Evaluation of the NucliSens EasyQ assay in HIV-1-infected individuals in South Africa.

We compared the performance of the NucliSens EasyQ assay (bioMerieux) combined with the manual NucliSens miniMag extraction methodology to the Roche Cobas Ampliprep/Standard Amplicor Monitor methodology (Roche Diagnostics) for HIV-1 RNA quantitation in HIV-1-infected individuals in South Africa. Plasma samples (284) from HIV sero-positive patients at different stages of infection were analyzed. The distribution of results was typical of the clinical samples received at the laboratory where 20% have viral load results <400 copies/ml (2.6 log) and 18% have viral load results >750000 copies/ml (5.8 log) using the Roche Amplicor Monitor standard assay. All statistical analyses were performed using log10-transformed values for all the variables in the analyses, i.e. log10EasyQIU/ml, and log10RNA (log10 copies/ml, Amplicor). Roche values were converted from RNA copies per ml to IU/ml by multiplying the Roche value by 0.51. HIV RNA levels quantitated by the NucliSens EasyQ assay correlated significantly with those of the Roche Cobas Amplicor Monitor assay (r=0.874, p<0.0001). Reproducibility of the NucliSens EasyQ assay in the log6IU range yielded CV variance of 1.3-2.84% for two well-trained technologists. In addition, a retrospective evaluation of the performance of the NucliSens EasyQ assay in 102 runs (2448) samples was conducted in the laboratory over a 4-month interval. Factors considered during this evaluation included time taken to perform the assay, volume requirements, number of required repeats, potential for contamination.

In vitro effects of thawing fresh-frozen plasma at various temperatures.

Thawing fresh-frozen plasma (FFP) in South Africa is uncontrolled because the plasma is issued frozen from the blood bank and thawing techniques and temperatures are at the discretion of the clinician. Following anecdotal reports of disseminated intravascular coagulation (DIC) developing in patients who received FFP thawed in an uncontrolled manner, the effects of various thawing temperatures on coagulation parameters were studied. Fifteen adult units of FFP were each divided into 4 satellite units by the South African Blood Transfusion Service before freezing at -25 degrees C. These bags were then defrosted in a waterbath at 22 degrees C, 37 degrees C, 45 degrees C and 60 degrees C, respectively, and removed as soon as they had thawed. Samples were collected for measurement of International Normalized Ratio (INR), prothrombin time (PT), partial thromboplastin time (PTT), fibrinogen, and D-dimers. These tests were done according to standard operating procedures. FFP samples were also used in place of agonist in platelet aggregation studies to assess whether the FFP could induce platelet aggregation. Results were analyzed with the percentage similarity model. Using this method the percentage similarity (%sim) of each bag thawed at each temperature with the same donor's bag thawed at 37 degrees C was calculated. The mean, standard deviation, and percentage coefficient of variation of the percentage similarities were then derived. Data sets were also compared using the Wilcoxon test. The fibrinogen values remained stable at 22-45 degrees C (%sim = 97-99%) while there was a significant decrease in fibrinogen levels at 60 degrees C compared with 37 degrees C (p<0.001, %sim = 75%). INR and PTT values were highest in the bags thawed at 60 degrees C (%sim = 114% and 110%, respectively) with the difference between the INR levels at 60 degrees C compared with 37 degrees C showing statistical significance (p<0.05). D-dimers were high at all temperatures tested with widely ranging results at each temperature. The FFP did not induce platelet aggregation at any of the thawing temperatures. In summary, INR and PTT values increase at a thawing temperature of 60 degrees C compared with 37 degrees C. D-dimers are elevated in thawed FFP. Fibrinogen levels are markedly decreased in FFP thawed at 60 degrees C compared with that thawed at 37 degrees C. FFP should be thawed at 37 degrees C in a strictly controlled environment.

Effect of removal of duplicate isolates on cumulative susceptibility reports.

The objective of our study is to assess the impact of different methods of duplicate isolate removal on cumulative susceptibility reports. Over a 1-year period, we studied the effect of 3 methods of duplicate isolate removal on the cumulative percentage susceptibility of 9 Gram-negative bacilli to 15 antimicrobials. Raw data from which no duplicate isolates were removed (NR) were generated by the Sensititre breakpoint susceptibility testing system. D3 and D7 were methods of duplicate isolate removal defined as follows: same patient, bacterial species, irrespective of susceptibility within either three (D3) or seven (D7) calendar days of the date of the previous culture. The third method evaluated was an algorithm utilized by Cerner, a laboratory management program that defines duplicate isolates as follows: same patient, bacterial species, and NCCLS susceptibility category to an individual antimicrobial. Differences in percentage susceptibility between the three methods of duplicate isolate removal and NR were assessed. The number of isolates studied ranged from 80 (E. aerogenes) to 681 (P. aeruginosa). Of the methods of duplicate isolate removal, the highest percentage susceptibility occurred most frequently with Cerner followed by D7 and D3. Differences in percentage susceptibility between methods of removal and NR ranged from -11 to 25%, -5 to 8%, and -3 to 10%, with Cerner, D3, and D7, respectively. The percentage susceptibility was at least 5% higher than NR with a method of removal for 15 individual organism/antimicrobial combinations in which susceptibility was > or = 70% by at least one of the methods. These occurred most frequently with Enterobacter species and Cerner. Although there is no consensus on the ideal method of duplicate isolate removal, one should be cognizant that these manipulations may produce different cumulative susceptibility reports.

CD4 cell monitoring for HIV/AIDS: old options; new insights.

Cost effective solutions are needed for laboratory monitoring that do not compromise on quality but address costs. Current recommended methods of CD4+ T cell enumeration are complex and costly. Monitoring typically utilises viral load assessment (PCR based) and CD4+ T cell counting to assess disease progression and response to therapy in HIV/AIDS. This paper reviews CD4 testing with the focus on different methods of CD4+ T cell enumeration including state-of-the-art flow cytometric testing, the advantages and disadvantages of these systems and quality control. Lastly, recent new work undertaken at the University of the Witwatersrand is discussed that addresses the problems of cost and precision and accuracy of CD4 T cell testing.

Studies of c-jun oncogene expression in human breast using a new monoclonal antibody, NCL-DK4.

A new monoclonal antibody to human c-jun oncoprotein, designated NCL-DK4, has been produced. NCL-DK4 has been proved to be highly effective for use on formalin-fixed, paraffin-embedded tissues, enabling the study of c-jun expression at a cellular level in both normal and neoplastic human tissues. The expression of c-jun oncogene has been examined in normal, benign, and malignant breast tissues, and c-jun-specific immunoreactivity in carcinomas has been related to histological type, tumour grade, c-erbB-2, oestrogen receptor, progesterone receptor, and epidermal growth factor receptor expression. Normal and benign breast tissues showed c-jun-specific immunostaining, which was weaker and in fewer cells compared with the c-jun immunoreactivity observed in breast carcinomas. No relationship was found between the degree of immunostaining and the extent of proliferative changes in benign breast tissues. Ninety per cent of all breast carcinomas studied showed c-jun-specific nuclear staining. There were no statistically significant differences in the intensity of c-jun immunoreactivity among grade I, II, and III infiltrating ductal carcinomas. There was no significant relationship between c-jun oncoprotein expression and c-erbB-2, oestrogen, progesterone, and epidermal growth factor receptor immunoreactivity.

Wipe tests to assess 99Tcm surface contamination: effects of surface type, swab type and chemical form.

The efficacy of wipe tests for assessing 99Tcm surface contamination was measured. Four types of surface were contaminated with four radiopharmaceuticals and wiped using four types of swab. The fraction of activity removed was measured by direct monitoring for each combination of surface, pharmaceutical and swab. The results obtained showed that wipe tests are neither accurate nor precise. Observed wipe efficacies differed greatly from the customary assumption that 10% of the activity is removed by wiping. Detergent soaked swabs gave a mean efficacy of 40%, although with considerable variation (coefficient of variation 49%). Using these swabs alone the surface type affected efficacy by almost a factor three (floor tile mean efficacy 20%, plastic laminate mean efficacy 57%). In principle this effect might be compensated for by using correction factors according to the surface being swabbed. However, the pharmaceutical type will generally be unknown, and this also affected efficacy by almost a factor of two (eluate mean efficacy 29%, macroaggregated albumin 53%). Overall the results suggest that wipe tests can be used to detect contamination but are unreliable for quantification.

A new technique for xenon-133 ventilation imaging in the diagnosis of pulmonary embolism.

A new approach to ventilation imaging of the lung using 133Xe in patients with suspected pulmonary embolism is described. Three inhalation images are obtained in the postero-anterior, right posterior oblique and left posterior oblique projections, respectively, and each image contains 140-180 kcounts. The technique is quick and simple and needs neither an oxygen supply nor a spirometer. It combines the multiple views characteristic of krypton imaging with the ease of availability of xenon.

Examining the interaction effects of coping style and brief interventions in the treatment of postsurgical pain.

The present study sought to ameliorate two major deficiencies in the literature on treating response to surgery, viz., the failure to compare clearly delineated treatments, alone and in combination; and the failure to examine treatment X coping style interactions. Information imparting and brief relaxation were examined in this study as they interacted with an avoidance-sensitization coping style. No differences were found between treatments or coping styles. Sensitizers, on the other hand, were found to profit most from the relaxation training. Avoiders appeared to do well when they were left alone. The interaction effect was demonstrated for both self-report measures of pain and a behavioral measure of potency of medications ingested. The effects on self-report of pain were more evident on the second postsurgical day than on the fourth postsurgical day. The results indicate that brief relaxation training, often the only kind available to the medical psychologist dealing with surgical patients, is best confined to patients with a sensitizing coping style. Further, the results of this study, in conjunction with a reanalysis of previous studies, cast considerable doubt on information imparting when presented alone as a viable technique for reducing the distress consequent on surgery.

Preoperative predictors of postoperative pain.

This study attempted to predict postoperative pain from preoperative level of anxiety and the amount of information patients possessed regarding their surgery. Pain was assessed via the McGill Pain Questionnaire (MPQ) and a measure of pain complaints--number of analgesics taken. High levels of state anxiety and a high degree of information predicted the Present Pain Intensity measured of the MPQ, but did not predict the Pain Rating Index portion of the MPQ. The number of analgesics taken was predicted from the amount of information but not the level of presurgical anxiety. Biographical variables were unrelated to postoperative pain. The results were discussed in terms of State-Trait Anxiety theory, Janis' curvilinear prediction model and a contextual perspective of information imparting.