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1.  Stability of Extemporaneously Prepared Lansoprazole Suspension at Two Temperatures 
OBJECTIVE
The purpose of this study was to examine the stability of a generic lansoprazole product in a 3 mg/mL sodium bicarbonate suspension under room temperature and refrigerated conditions.
METHODS
Lansoprazole suspensions (3 mg/mL) were prepared in triplicate using an 8.4% sodium bicarbonate vehicle for each storage condition (room temperature and refrigerated). During 1 month, samples from each replicate were periodically removed and analyzed for lansoprazole concentration by liquid chromatography–tandem mass spectrometry (LC-MS/MS). Each sample was spiked with 10 mg/L omeprazole to serve as the internal standard. A positive electrospray LC-MS/MS method was validated over the calibration range of 5 to 25 mg/L using Food and Drug Administration Guidance. The identities of the analyte and internal standard in the samples were verified by monitoring the MS/MS transitions of m/z 370 to m/z 252 and m/z 346 to m/z 198 for lansoprazole and omeprazole, respectively. Additionally, the pH of the suspensions was monitored throughout the study.
RESULTS
The stability of lansoprazole in the oral sodium bicarbonate suspension under refrigeration is compromised prior to what has been previously reported in the literature. Samples kept at room temperature lost >10% of the lansoprazole after 48 hours compared with the refrigerated samples, which maintained integrity up to 7 days. No statistically significant difference was found between the pH of the room temperature and refrigerated suspension samples, indicating that this factor is not the cause for the differences in stability at these two conditions.
CONCLUSIONS
This study suggests that the extemporaneously compounded lansoprazole oral suspension prepared in 8.4% sodium bicarbonate should not be stored in plastic oral syringes longer than 48 hours at room temperature and no longer than 7 days when refrigerated. These data indicate an expiration time earlier than that previously reported for the refrigerated product (14 days).
doi:10.5863/1551-6776-18.2.122
PMCID: PMC3668940  PMID: 23798906
drug stability; lansoprazole; suspensions
2.  Time-Dependent Effect of Refrigeration on Viscosity and Conversion Kinetics of Dental Adhesive Resins 
European Journal of Dentistry  2010;4(2):150-155.
Objectives:
This study evaluated the effect of refrigeration at 4°C and post-refrigeration times (immediate, 5, 10, 15, or 20 min) on the viscosity and conversion kinetics of adhesive bonding resins.
Methods:
Scotchbond Dual-Cure (3M ESPE) and Clearfil SE Bond (Kuraray) were tested. Control samples were kept at 25°C for 24 h. At each post-refrigeration time, the temperature was checked with a K-type thermocouple. Viscosity measurements as a function of temperature were performed using a cone-plate viscometer. Real-time polymerization was monitored by infrared spectroscopy. Degree of conversion (DC) was calculated for each second during polymerization, and the rate of polymerization analyzed. Data were separately submitted to two-way ANOVA and Tukey’s test (P<.05).
Results:
Clearfil presented faster increase in temperature after exposure to room temperature than Scotchbond. A continuous decrease in viscosity (Pa.s) was observed for both Scotchbond (0.49, 0.34, 0.30, 0.26, 0.23, 0.23) and Clearfil (0.38, 0.37, 0.34, 0.25, 0.24, 0.22). For Scotchbond, higher final DC was detected for the control (62.7%) compared with the immediate (53.3%) and 5 min (54.7%) groups. For Clearfil, the control sample (81.4%) showed higher DC than all refrigerated groups (68.8–69.5%). Clearfil always showed significantly higher DC than Scotchbond.
Conclusions:
Refrigeration presented a significant time- and material-dependent effect on the viscosity and polymerization kinetics of the bonding resins. Under clinical conditions, adhesive agents should be removed from the refrigerator at least 20 min before being used.
PMCID: PMC2853827  PMID: 20396445
Bonding resins; Degree of conversion; Polymerization kinetics; Refrigeration; Viscosity
3.  Stability of Ziprasidone Mesylate in an Extemporaneously Compounded Oral Solution 
OBJECTIVES
To formulate a liquid preparation of ziprasidone in a convenient concentration to allow dosing of less than 20 mg and of sufficient chemical and physical stability to enable an entire prescription or course of treatment to be prepared in a single batch.
METHODS
Geodon for injection (ziprasidone mesylate), 20 mg/mL, was diluted to 2.5 mg/mL in a commercially available sugar-free and alcohol-free, flavored syrup and stored at room temperature under ambient fluorescent light illumination, at room temperature in darkness, and under refrigeration. The ziprasidone content was measured in samples at various time intervals using a stability-indicating high-performance liquid chromatographic method.
RESULTS
When refrigerated, the ziprasidone syrup that was compounded in a commercially available, sugar-free and alcohol-free vehicle maintained at least 90% of stated potency for at least 6 weeks. Samples stored under other conditions were less stable, underscoring the manufacturer's labeling regarding refrigerated storage of the reconstituted injection.
CONCLUSIONS
The findings suggest that chemical and physical stability are maintained for 2 weeks under refrigeration, allowing the convenience of compounding for the long-term needs of a particular patient, rather than daily compounding. The only storage condition we recommend is refrigeration at 5°C.
PMCID: PMC3018175  PMID: 22477804
extemporaneous compounding; high performance liquid chromatography; oral solution; stability; ziprasidone
4.  Chiral Stability of an Extemporaneously Prepared Clopidogrel Bisulfate Oral Suspension 
OBJECTIVES
The purpose of this study was to evaluate the chiral stability of clopidogrel bisulfate in an extemporaneously compounded oral suspension for a period of 60 days.
METHODS
A 5 mg/mL oral suspension of clopidogrel bisulfate was prepared from commercially available Plavix tablets. The clopidogrel suspension was then evenly divided between two light-resistant prescription bottles and stored either under refrigeration (4°C) or at room temperature (25°C). Samples were drawn from the stored suspensions immediately after preparation and on days 7, 14, 28, and 60. Samples were subsequently analyzed at each time point by high-performance liquid chromatography using a reversed-phase column, with chemical stability defined as the retention of at least 90% of the initial intact clopidogrel concentration measured. To determine the chiral stability of the suspension, samples were also analyzed by high-performance liquid chromatography using a chiral column to investigate possible enantiomeric inversion. Chiral stability was defined as the retention of at least 90% of the initial concentration of the suspension as the S-enantiomer, the active moiety of Plavix.
RESULTS
Regardless of storage conditions, the oral suspension of clopidogrel retained at least 98% of the active S-enantiomer for 60 days after preparation. Compared with the clopidogrel suspension stored in the refrigerator, more chiral inversion was noted in the clopidogrel suspension stored at room temperature.
CONCLUSIONS
Our investigation of chiral stability indicates that a 5 mg/mL clopidogrel oral suspension stored under refrigeration and at room temperature maintains chiral stability as the active S-enantiomer.
doi:10.5863/1551-6776-19.1.25
PMCID: PMC3998964  PMID: 24782688
chiral; clopidogrel bisulfate; compounded oral suspension; enantiomers; high-performance liquid chromatography
5.  The effects of storage conditions on viability of Clostridium difficile vegetative cells and spores and toxin activity in human faeces 
Journal of Clinical Pathology  2003;56(2):126-128.
Aims:Clostridium difficile is a common nosocomial pathogen and as such diagnostic and research methods may necessitate storage of faecal specimens for long periods, followed by subsequent re-examination. This study investigated the effects of storage conditions upon the viability of this organism and its toxin.
Methods: Three genotypically distinct strains of C difficile (two clinical isolates including the UK epidemic strain, and an environmental isolate) were grown anaerobically at 37°C for 72 hours in a pool of five faecal emulsions. Aliquots of each emulsion were stored at either −20°C (frozen) or 4°C (refrigerated). Emulsions were assayed for viable cells, spores, and cytotoxin titre before storage and at days 1, 3, 5, 7, 14, 28, and 56. An aliquot of each emulsion was also removed, assayed, and replaced in storage at each time point to investigate the effects of multiple freezing/refrigeration/thawing.
Results: Neither storage temperature nor multiple cycles of refrigeration/freezing and thawing adversely affected the viability of C difficile vegetative cells or spores. Single and multiple exposures of samples to 4°C had little effect upon the C difficile toxin titre. Toxin titres of multiply frozen and thawed faeces became significantly lower than for refrigerated faeces (p < 0.01) by day 5 of the experiment in two of the three strains, and in all strains by day 28. Toxin titres of singly frozen faeces became significantly lower than for refrigerated faeces (p < 0.01) by day 56 of the experiment in two of the three strains.
Conclusion: Storage temperature and multiple cycles of freezing (refrigeration)/thawing had minimal effects upon the viability of C difficile or its spores. Storage at 4°C has no discernible effect on C difficile cytotoxin. However, storage at −20°C has a detrimental effect upon C difficile cytotoxin, and multiple cycles of freezing and thawing may further adversely effect toxin titres.
PMCID: PMC1769877  PMID: 12560391
Clostridium difficile; storage temperature; viability; toxin degradation
6.  Stability of Diluted Adenosine Solutions in Polyolefin Infusion Bags 
Hospital Pharmacy  2013;48(6):484-488.
Background:
Intravenous or intracoronary adenosine is used in the cardiac catherization lab to achieve maximal coronary blood flow and determine fractional flow reserve.
Objective:
To determine the stability of adenosine 10 and 50 µg/mL in either 0.9% sodium chloride injection or 5% dextrose injection in polyolefin infusion bags stored at 2 temperatures, refrigeration (2°C-8°C) or controlled room temperature (20°C-25°C).
Methods:
Adenosine 10 µg/mL and 50 µg/mL solutions were prepared in 50 mL polyolefin infusion bags containing 0.9% sodium chloride injection or 5% dextrose injection and stored at controlled room temperature or under refrigeration. Each combination of concentration, diluent, and storage was prepared in triplicate. Samples were assayed using stability-indicating, reversed-phase high-performance liquid chromatography immediately at time 0 and at 24 hours, 48 hours, 7 days, and 14 days. Stability was defined as retaining 90% to 110% of the initial adenosine concentration. The samples were also visually inspected against a light background for clarity, color, and the presence of particulate matter.
Results:
After 14 days, all samples retained 99% to 101% of the initial adenosine concentration. No considerable change in pH or visual appearance was noted. The stability data indicated no significant loss of drug due to chemical degradation or physical interactions during storage.
Conclusion:
Adenosine solutions of 10 and 50 µg/mL were stable for at least 14 days in 50 mL polyolefin infusion bags of 0.9% sodium chloride injection or 5% dextrose injection stored at controlled room temperature and refrigerated conditions.
doi:10.1310/hpj4806-484
PMCID: PMC3839492  PMID: 24421510
adenosine; concentration; fractional flow reserve; stability
7.  Impact of Round-the-Clock, Rapid Oral Fluid HIV Testing of Women in Labor in Rural India 
PLoS Medicine  2008;5(5):e92.
Background
Testing pregnant women for HIV at the time of labor and delivery is the last opportunity for prevention of mother-to-child HIV transmission (PMTCT) measures, particularly in settings where women do not receive adequate antenatal care. However, HIV testing and counseling of pregnant women in labor is a challenge, especially in resource-constrained settings. In India, many rural women present for delivery without any prior antenatal care. Those who do get antenatal care are not always tested for HIV, because of deficiencies in the provision of HIV testing and counseling services. In this context, we investigated the impact of introducing round-the-clock, rapid, point-of-care HIV testing and counseling in a busy labor ward at a tertiary care hospital in rural India.
Methods and Findings
After they provided written informed consent, women admitted to the labor ward of a rural teaching hospital in India were offered two rapid tests on oral fluid and finger-stick specimens (OraQuick Rapid HIV-1/HIV-2 tests, OraSure Technologies). Simultaneously, venous blood was drawn for conventional HIV ELISA testing. Western blot tests were performed for confirmatory testing if women were positive by both rapid tests and dual ELISA, or where test results were discordant. Round-the-clock (24 h, 7 d/wk) abbreviated prepartum and extended postpartum counseling sessions were offered as part of the testing strategy. HIV-positive women were administered PMTCT interventions. Of 1,252 eligible women (age range 18 y to 38 y) approached for consent over a 9 mo period in 2006, 1,222 (98%) accepted HIV testing in the labor ward. Of these, 1,003 (82%) women presented with either no reports or incomplete reports of prior HIV testing results at the time of admission to the labor ward. Of 1,222 women, 15 were diagnosed as HIV-positive (on the basis of two rapid tests, dual ELISA and Western blot), yielding a seroprevalence of 1.23% (95% confidence interval [CI] 0.61%–1.8%). Of the 15 HIV test–positive women, four (27%) had presented with reported HIV status, and 11 (73%) new cases of HIV infection were detected due to rapid testing in the labor room. Thus, 11 HIV-positive women received PMTCT interventions on account of round-the-clock rapid HIV testing and counseling in the labor room. While both OraQuick tests (oral and finger-stick) were 100% specific, one false-negative result was documented (with both oral fluid and finger-stick specimens). Of the 15 HIV-infected women who delivered, 13 infants were HIV seronegative at birth and at 1 and 4 mo after delivery; two HIV-positive infants died within a month of delivery.
Conclusions
In a busy rural labor ward setting in India, we demonstrated that it is feasible to introduce a program of round-the-clock rapid HIV testing, including prepartum and extended postpartum counseling sessions. Our data suggest that the availability of round-the-clock rapid HIV testing resulted in successful documentation of HIV serostatus in a large proportion (82%) of rural women who were unaware of their HIV status when admitted to the labor room. In addition, 11 (73%) of a total of 15 HIV-positive women received PMTCT interventions because of round-the-clock rapid testing in the labor ward. These findings are relevant for PMTCT programs in developing countries.
Nitika Pant Pai and colleagues report the results of offering a round-the-clock rapid HIV testing program in a rural labor ward setting in India.
Editors' Summary
Background.
Since the first reported case of AIDS (acquired immunodeficiency syndrome) in 1981, the number of people infected with the human immunodeficiency virus (HIV), which causes AIDS, has risen steadily. Now, more than 33 million people are infected, almost half of them women. HIV is most often spread through unprotected sex with an infected partner, but mother-to-child transmission (MTCT) of HIV is also an important transmission route. HIV-positive women often pass the virus to their babies during pregnancy, labor and delivery, and breastfeeding, if nothing is done to prevent viral transmission. In developed countries, interventions such as voluntary testing and counseling, safe delivery practices (for example, offering cesarean delivery to HIV-positive women), and antiretroviral treatment of the mother during pregnancy and labor and of her newborn baby have minimized the risk of MTCT. In developing countries, the prevention of MTCT (PMTCT) is much less effective, in part because pregnant women often do not know their HIV status. Consequently, in 2007, nearly half a million children became infected with HIV mainly through MTCT.
Why Was This Study Done?
In many developing countries, women do not receive adequate antenatal care. In India, for example, nearly half the women living in rural areas do not receive any antenatal care until they are in labor. This gives health care providers very little time in which to counsel women about HIV infection, test them for the virus, and start interventions to prevent MTCT. Furthermore, testing pregnant women in labor for HIV and counseling them is a challenge, particularly where resources are limited. In this study, therefore, the researchers investigate the feasibility and impact of introducing round-the-clock, rapid HIV testing and counseling in a busy labor ward in a rural teaching hospital in Sevagram, India.
What Did the Researchers Do and Find?
Women admitted to the labor ward between January and September 2006 were offered two rapid HIV tests—one that used a saliva sample and the other that used blood taken from a finger prick. Blood was also taken from a vein for conventional HIV testing. All the women were given a 15-minute counseling session about how HIV is transmitted, the importance of HIV testing, and information on PMTCT before their child was born (prepartum counseling), and a longer postpartum counseling session. HIV-positive women were given a cesarean delivery where possible and antiretroviral drug treatment to reduce MTCT. 1,222 women admitted to the labor ward during the study period (1,003 of whom did not know their HIV status) accepted HIV testing. Of 15 study participants who were HIV positive, 11 learnt of their HIV status in the labor room. Two babies born to these HIV-positive women were HIV positive and died within a month of delivery; the other 13 babies were HIV negative at birth and at 1 and 4 months after delivery. Finally, the rapid HIV tests missed only one HIV-positive woman (no false-positive results were given), and the time from enrolling a woman into the study through referring her for PMTCT intervention where necessary averaged 40–60 minutes.
What Do These Findings Mean?
These findings show the feasibility and positive impact of the introduction of round-the-clock pre- and postpartum HIV counseling and rapid HIV testing into a busy rural Indian labor ward. Few of the women entering this ward knew their HIV status previously but the introduction of these facilities in this setting successfully informed these women of their HIV status. In addition, the round-the-clock counseling and testing led to 11 women and their babies receiving PMTCT interventions who would otherwise have been missed. These findings need to be confirmed in other settings and the cost-effectiveness and sustainability of this approach for the improvement of PMTCT in developing countries needs to be investigated. Nevertheless, these findings suggest that round-the-clock rapid HIV testing might be an effective and acceptable way to reduce MTCT of HIV in many developing countries.
Additional Information.
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0050092.
Read a related PLoS Medicine Perspective article
Information is available from the US National Institute of Allergy and Infectious Diseases on HIV infection and AIDS and on HIV infection in women
HIV InSite has comprehensive information on all aspects of HIV/AIDS
Women, Children, and HIV provides extensive information on the prevention of mother-to-child transmission of HIV in developing countries
Information is available from Avert, an international AIDS charity, on HIV and AIDS in India, on women, HIV, and AIDS, and on HIV and AIDS prevention, including the prevention of mother-to-child transmission
AIDSinfo, a service of the US Department of Health and Human Services provides health information for HIV-positive pregnant women (in English and Spanish)
doi:10.1371/journal.pmed.0050092
PMCID: PMC2365974  PMID: 18462011
8.  Enhanced shear-induced platelet aggregation due to low-temperature storage 
Transfusion  2012;53(7):1520-1530.
BACKGROUND
Refrigeration of platelets (PLTs) offers an attractive alternative to the currently practiced storage at room temperature since it may mitigate problems associated with bacterial contamination and extend storage lifetime. Refrigeration causes a number of biophysical and biochemical changes in PLTs and decreases PLT circulation time in vivo. However, the effect of refrigeration on PLT hemostatic functions under physiologic and pathophysiologic shear conditions has not been adequately characterized.
STUDY DESIGN AND METHODS
Washed PLTs prepared from either fresh PLT-rich plasma (PRP) or PRP stored at 4°C for 2 days was mixed with exogenous von Willebrand factor (VWF) and fibrinogen and sheared in a cone-and-plate viscometer. PLT aggregation, activation, and VWF binding after shear and glycoprotein (GP) Ibα receptor expression and ristocetin-induced PLT agglutination were measured.
RESULTS
PLTs stored at 4°C for 2 days aggregated significantly more than fresh PLTs particularly at high shear rates (10,000/sec), and this increase was independent of PLT concentration or suspension viscosity. Further, refrigerated PLTs showed a greater increase in GP Ibα–dependent PLT activation under shear and also bound more VWF than fresh PLTs. However, the GP Ibα expression levels as measured by three different antibodies were significantly lower in refrigerated PLTs than in fresh PLTs, and refrigeration resulted in a modest decrease in ristocetin-induced PLT agglutination.
CONCLUSION
The combined results demonstrate that refrigeration increases PLT aggregation under high shear, but not static, conditions and also increases shear-induced VWF binding and PLT activation. Clinically, enhanced shear-induced PLT aggregation due to low temperature storage may be a beneficial strategy to prevent severe bleeding in trauma.
doi:10.1111/j.1537-2995.2012.03917.x
PMCID: PMC4321779  PMID: 23043289
9.  Lack of Mucosal Immune Reconstitution during Prolonged Treatment of Acute and Early HIV-1 Infection 
PLoS Medicine  2006;3(12):e484.
Background
During acute and early HIV-1 infection (AEI), up to 60% of CD4+ T cells in the lamina propria of the lower gastrointestinal (GI) tract are lost as early as 2–4 wk after infection. Reconstitution in the peripheral blood during therapy with highly active antiretroviral therapy (HAART) is well established. However, the extent of immune reconstitution in the GI tract is unknown.
Methods and Findings
Fifty-four AEI patients and 18 uninfected control participants underwent colonic biopsy. Forty of the 54 AEI patients were followed after initiation of antiretroviral therapy (18 were studied longitudinally with sequential biopsies over a 3-y period after beginning HAART, and 22 were studied cross sectionally after 1–7 y of uninterrupted therapy). Lymphocyte subsets, markers of immune activation and memory in the peripheral blood and GI tract were determined by flow cytometry and immunohistochemistry. In situ hybridization was performed in order to identify persistent HIV-1 RNA expression. Of the patients studied, 70% maintained, on average, a 50%–60% depletion of lamina propria lymphocytes despite 1–7 y of HAART. Lymphocytes expressing CCR5 and both CCR5 and CXCR4 were persistently and preferentially depleted. Levels of immune activation in the memory cell population, CD45RO+ HLA-DR+, returned to levels seen in the uninfected control participants in the peripheral blood, but were elevated in the GI tract of patients with persistent CD4+ T cell depletion despite therapy. Rare HIV-1 RNA–expressing cells were detected by in situ hybridization.
Conclusions
Apparently suppressive treatment with HAART during acute and early infection does not lead to complete immune reconstitution in the GI mucosa in the majority of patients studied, despite immune reconstitution in the peripheral blood. Though the mechanism remains obscure, the data suggest that there is either viral or immune-mediated accelerated T cell destruction or, possibly, alterations in T cell homing to the GI tract. Although clinically silent over the short term, the long-term consequences of the persistence of this lesion may emerge as the HIV-1–infected population survives longer owing to the benefits of HAART.
Despite early initiation of anti-HIV therapy, loss of T cells in the gastrointestinal mucosa persisted for years in most members of a clinical cohort identified early after HIV-1 infection.
Editors' Summary
Background.
AIDS causes disease by inactivating the body's immune response against infection. The AIDS virus (HIV) is most active against the white blood cells called T lymphocytes, particularly the CD4 T lymphocytes, which recognize infection and activate other cells of the immune system to fight it. In what was formerly believed to be a gradual process, HIV infection is now known to deplete a subset of the body's CD4 lymphocytes, called memory cells, quite rapidly—over only a few days—within a few weeks after a person becomes infected with the AIDS virus. This was not known until recently because researchers were counting CD4 cells only in blood, while a majority of the memory lymphocytes are located in and around the digestive system. It is these intestinal memory lymphocytes that are rapidly wiped out, while those in the blood fall much more gradually, usually over several years. Few studies of mucosal lymphocytes have been done in humans because such studies require biopsies of the intestinal lining (mucosa).
Why Was This Study Done?
Although CD4 cells in the blood can return and remain at normal levels when HIV infection is treated with antiviral drugs, it has been unclear as to whether the mucosal CD4 cells return as well. People who begin treatment as soon as possible after becoming infected with HIV might seem to have the best chance of regaining their mucosal immunity, compared to those who wait until the CD4 cells in their blood have fallen, which is a generally accepted reason to start medication for HIV. Therefore, the researchers wanted to see whether people who start treatment early after becoming infected with HIV might experience restoration of their mucosal immunity over time and, if so, what kinds of lymphocytes would return.
What Did the Researchers Do and Find?
The researchers studied people who started treatment for HIV within a few weeks to months of becoming infected and who then remained on treatment. Some volunteers underwent biopsies of the intestinal mucosa before starting treatment and then at various points from 1 y until as long as 3 y after infection. Others volunteered for biopsy only one time, anywhere from less than 1 y to 7 y following treatment. The biopsy specimens were examined under the microscope and with a technique called flow cytometry using specific staining methods to assess their structure and functional characteristics. Results were compared to biopsies from a group of HIV-uninfected volunteers.
The researchers found that the percentage of CD4 lymphocytes dropped much lower in the intestinal mucosa than in blood during early infection and then, unlike in blood, remained low even after several years of treatment for HIV. In the microscope images, they found that mucosal CD4 cells were lost mostly from regions of active battle against invading germs, rather than from “training sites” for new CD4 cells. Over time, only 30% of the volunteers showed return of CD4 cells to normal levels in these active sites.
Unlike T lymphocytes in the blood, which tend to return to a resting state after HIV is treated, the T lymphocytes in the intestinal mucosa tended to persist in an activated state despite HIV treatment, even though only a tiny fraction of these cells were found to be infected with HIV. A high level of activation of mucosal lymphocytes soon after infection was found to predict poor restoration of mucosal CD4 cells over time.
What Do These Findings Mean?
These experiments confirm that studying easily obtained blood lymphocytes provides only a limited view of how HIV affects the immune system as a whole. The finding that immune cells of the intestinal mucosa remain depleted and over-activated for years despite antiretroviral treatment raises the concern that over time this will result in clinical problems. Fortunately, this does not appear to be the case in most people currently being treated for HIV, some for as long as 10 y, but the results of this study suggest that we should remain vigilant for gastrointestinal problems resulting from impaired immunity over time. The finding that mucosal lymphocytes do appear to return to normal levels in a minority of volunteers is of interest, and suggests that early interventions to reduce activation of intestinal T cells (such as antimicrobial or immunomodulatory treatment) might be worth investigating in those recently infected with HIV. Finally, these results suggest that a vaccine to prevent HIV may need to stimulate immune responses that can act very quickly following infection, before the bulk of lymphocytes capable of countering the infection are lost, perhaps irreversibly.
Additional Information.
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0030484.
UCSF HIV InSite includes resources on HIV immunology and vaccine development
AIDS fact sheets and brochures from the US National Institute of Allergy and Infectious Diseases
Medline Plus article on acute HIV infection from the US National Library of Medicine
doi:10.1371/journal.pmed.0030484
PMCID: PMC1762085  PMID: 17147468
10.  Stability of Vancomycin 25 mg/mL in Ora-Sweet and Water in Unit-Dose Cups and Plastic Bottles at 4°C and 25°C 
Background:
Solutions of vancomycin for oral administration are not available commercially in Canada or the United States but are needed for patients who cannot swallow capsules.
Objective:
To evaluate the stability of vancomycin solutions stored in unit-dose cups and plastic bottles under refrigeration (4°C) and at room temperature (25°C) for up to 75 days.
Methods:
Vancomycin 25 mg/mL in Ora-Sweet vehicle and water (1:1 ratio by volume) was dispensed into opaque blue polyethylene unit-dose cups with aluminum seal (14 replicates) or amber plastic prescription bottles (6 replicates). Seven cups and 3 bottles were refrigerated (4°C), and the remainder of the containers were stored at room temperature (25°C). At the time of preparation and at 15, 30, 40, 50, 63, and 75 days, 3 aliquots were collected from one of the cups and from every bottle and were stored frozen (−85°C) until the time of analysis. Physical characteristics were evaluated at each time point, including measurement of pH and visual assessment of colour and precipitation. After thawing, the samples were analyzed in triplicate by a validated stability-indicating high-performance liquid chromatography method. A solution was considered stable if 90% of the initial concentration of vancomycin was maintained.
Results:
No notable changes in colour, taste, or pH were observed in vancomycin solutions stored in the unit-dose cups at 4°C or 25°C or in the plastic bottles stored at 4°C over the 75-day study period. Starting on day 63, a white precipitate was observed in the solutions stored in plastic bottles at 25°C, but there were no notable changes in taste or pH during the 75-day period. The 95% confidence interval of the slope of the curve relating concentration to time, determined by linear regression, indicated that vancomycin solutions stored in cups or bottles at 4°C would maintain at least 93.6% of the initial vancomycin concentration for 75 days and that solutions stored at 25°C would maintain at least 90.0% of the initial concentration for 30 days (cups) or 26 days (bottles), with 95% confidence.
Conclusions:
Vancomycin 25 mg/mL stored in unit-dose cups or plastic bottles at 4°C was stable for at least 75 days, whereas solutions stored in cups or bottles at 25°C are expected to be stable for 30 or 26 days, respectively.
PMCID: PMC2999368  PMID: 22479004
vancomycin; high-pressure liquid chromatography; stability; vancomycine; chromatographie liquide haute performance; stabilité
11.  Evaluation of a Newly Developed Lateral Flow Immunoassay for the Diagnosis of Cryptococcosis 
This study, evaluating the performance of a novel cryptococcal lateral flow immunoassay, shows that the assay performs as well as available diagnostic methods is economical, rapid, and easy to perform; and as such can be a point of care test in resource limited settings.
Background. Cryptococcosis is a common opportunistic infection of human immunodeficiency virus (HIV)–infected individuals mostly occurring in resource-limited countries. This study compares the performance of a recently developed lateral flow immunoassay (LFA) to blood culture and enzyme immunoassay (EIA) for the diagnosis of cryptococcosis.
Methods. Archived sera from 704 HIV-infected patients hospitalized for acute respiratory illness in Thailand were tested for cryptococcal antigenemia using EIA. All EIA-positive and a subset of EIA-negative sera were tested by LFA, with results recorded after 5 and 15 minutes incubation. Urine from patients with LFA- and EIA-positive sera was tested by LFA. Antigen results from patients with positive cryptococcal blood cultures were compared.
Results. Of 704 sera, 92 (13%) were positive by EIA; among the 91 EIA-positive sera tested by LFA, 82 (90%) and 87 (96%) were LFA positive when read after 5 and 15 minutes, respectively. Kappa agreement of EIA and LFA for sera was 0.923 after 5 minutes and 0.959 after 15 minutes, respectively. Two of 373 EIA-negative sera were LFA positive at both time points. Of 74 urine specimens from EIA-positive patients, 52 (70.3%) were LFA positive. EIA was positive in 16 of 17 sera from blood culture–positive patients (94% sensitivity), and all sera were positive by LFA (100% sensitivity).
Conclusions. A high level of agreement was shown between LFA and EIA testing of serum. The LFA is a rapid, easy-to-perform assay that does not require refrigeration, demonstrating its potential usefulness as a point-of-care assay for diagnosis of cryptococcosis in resource-limited countries.
doi:10.1093/cid/cir379
PMCID: PMC3148258  PMID: 21810743
12.  Stability of Cefazolin Sodium in Polypropylene Syringes and Polyvinylchloride Minibags 
Background:
Cefazolin is a semisynthetic penicillin derivative with a narrow spectrum of activity covering some gram-positive organisms and a few gram-negative aerobic bacteria.
Objective:
To determine the physical and chemical stability of cefazolin sodium reconstituted with sterile water for injection and stored in polypropylene syringes or diluted with either 5% dextrose in water (D5W) or 0.9% sodium chloride (normal saline [NS]) and stored in polyvinylchloride (PVC) minibags.
Methods:
Reconstituted solutions of cefazolin (100 or 200 mg/mL) were packaged in polypropylene syringes. More dilute solutions (20 or 40 mg/mL) were prepared in D5W or NS and packaged in PVC minibags. For each concentration–diluent–container combination, 3 containers were designated for each day of analysis (days 7, 14, 21, and 30). Containers were stored under refrigeration (5°C) with protection from light until the designated day of analysis, at which time one 5-mL sample was collected from each the designated container. The designated containers were then stored at room temperature (21°C to 25°C) with exposure to light for an additional 72 h, and additional samples were drawn. The samples were assayed using a validated, stability-indicating high-performance liquid chromatography method. The colour and clarity of the solutions, as well as their pH, were also monitored on each sampling day.
Results:
All samples remained clear for the duration of the study; they had a slight yellow colour that darkened over time, and there was an increase in pH. Solutions diluted with sterile water for injection and stored in polypropylene syringes retained at least 94.5% of the initial concentration after 30 days of refrigerated storage and at least 92.1% after an additional 72 h at room temperature with exposure to light. Samples diluted in D5W or NS and stored in PVC minibags retained at least 95.8% of the initial concentration after 30 days of refrigerated storage and at least 91.8% after an additional 72 h at room temperature with exposure to light.
Conclusions:
Cefazolin at various concentrations stored in polypropylene syringes or PVC minibags was stable for up to 30 days with storage at 5°C with protection from light, followed by an additional 72 h at 21°C to 25°C with exposure to light.
PMCID: PMC3161797  PMID: 22479065
cefazolin sodium; dextrose; normal saline; polypropylene syringes; polyvinylchloride minibags; stability; high-performance liquid chromatography; céfazoline sodique; dextrose; solution physiologique salée; seringues de polypropylène; minisacs de polychlorure de vinyle; stabilité; chromatographie liquide haute performance
13.  LEUCOCYTIC SECRETIONS 
In the present condition of the technique of cultivation of tissues, the only possible way of studying leucocytic secretions was to grow colonies of leucocytes in a medium of known properties and to examine the modifications of these properties under the influence of the living cells. The method was far from perfect, because the secretions were mixed with serum and accumulated for 48 hours in a medium where they probably underwent partial destruction. But an approximate idea of certain of the qualities of the secretions, although not of their quantity, could be derived from the experiments. In the fluids extracted from the cultures, we attempted to detect the presence of the leucocytic secretions through their physiological effects on homologous and foreign cells. Two kinds of substances were sought, those which act on homologous cells, and those which destroy foreign erythrocytes. The secretion by leucocytes of substances necessary to the nutrition of other cells was considered as probable long ago. Renaut thought that the main function of the white blood corpuscles was to bring to the fixed cells of the tissues the food material which they need. While the existence of physiological relations between leucocytes and tissue cells could be considered as almost certain, their nature had remained practically unknown. It was probable, however, that the substances secreted by leucocytes were analogous to the growth-activating and unstable substances which are found in embryonic tissues, leucocytes, and certain adult tissues. When connective tissue was aseptically inflamed, or when an aseptic peritoneal exudate contained many leucocytes, aqueous extracts of both connective tissue and peritoneal exudate were found to have acquired the power of stimulating cell proliferation. These experiments showed that leucocytes could bring to the tissues some activating substances. But it remained to be ascertained whether leucocytes, while they are alive, could secrete similar substances either spontaneously or under the stimulus of a foreign factor. Leucocytes are supposed to be, as is well know, the origin of the substances which protect the organism against infection. Although the problem of the origin of alexin and antibodies has been investigated by many experimenters, it is not yet completely solved. It was of interest, therefore, to ascertain whether leucocytic secretions could increase the natural hemolytic effect of hen serum on sheep or rabbit erythrocytes, and whether these secretions would become more active under the influence of a foreign protein. The substances which destroy foreign cells are not necessarily different from those which act on homologous cells. The word substance is used for simplicity of description and may be taken as meaning only a given property of an unknown substrate. A comparison was made of certain properties of sera extracted after 48 hours incubation from media containing leucocytes and from media containing no leucocytes. The serum from the leucocytic cultures was always found to be more favorable to the growth of homologous fibroblasts than the serum from the culture media incubated without leucocytes. The natural hemolytic power of the serum on sheep erythrocytes was found to be increased in about SO per cent of the experiments. In other experiments, we found that when two culture media free of cells were placed, one in an incubator at +38°C. and the other in a refrigerator at +5°C. for 48 hours, the serum from the incubated medium partly lost its hemolytic action on sheep or rabbit erythrocytes, while that from the refrigerated medium remained normal; at the same time, the inhibiting action of the incubated medium on homologous fibroblasts had increased very much. This effect of incubation indicates that certain unstable constituents of serum are destroyed by heat. Then the changes found in the properties of the serum from cultures of leucocytes are due to the fraction of the activating substances which has not been destroyed by incubation at 38°C. A quantitative study of the secretions is, therefore, impossible with the present technique, which can furnish only qualitative indications about the substances set free by the leucocytes. We have ascertained also whether a medium containing leucocytes and kept in the refrigerator undergoes any change under the influence of the cells while they are in a condition of latent life. Gabritschewski dishes with and without leucocytes were placed in a refrigerator at a temperature of about +5°C. After 48 hours, the hemolytic power on sheep erythrocytes of the serum from the leucocytic cultures had increased slightly and its inhibiting action on the growth of homologous fibroblasts had decreased. Then certain substances favorable to the growth of homologous cells and toxic for heterologous cells were diffused by the leucocytes into their medium. But the action of these substances was weaker than in the case of the cultures kept in the incubator. This experiment showed that leucocytes under certain conditions diffuse alexin or natural hemolysins which originate from them at the same time as the substances which activate homologous cells. In other experiments, although leucocytes were frozen at –10°C., treated with distilled water, or extracted with saline solution, they did not yield any hemolysin. To summarize: Leucocytes, cultivated in plasma, always secreted substances which increased the rate of growth of homologous cells. Less frequently, they set free substances which hemolyzed foreign erythrocytes. The growth-promoting substances are analogous to those contained in embryonic tissues, and probably represent some of the foodstuffs brought to fixed tissue cells by leucocytes. They may possess the function of rejuvenating cells which have ceased to multiply when the cicatrization of a wound or the repair of a fracture requires a resumption of tissue activity. According to this hypothesis, the leucocytes brought to the surface of a wound by the process of inflammation would not only oppose bacterial invasion, but also bring to the tissues the material necessary to cell multiplication. It seems that in some cases regeneration is started by substances brought to the tissues by other cells. Loeb thinks that in Tubularia, when endodermic cells gather at the end where a new polyp is about to be formed, the substances given off by these cells are responsible for polyp formation.6 There may be an analogy between this phenomenon and the secretion by leucocytes of growth-activating substances at the surface of a wound. If we assume that leucocytes in vivo set free their secretions in the blood stream, certain variations of the growth-inhibiting action of normal serum can be better understood. The rate of proliferation of homologous fibroblasts is much slower in the serum of an old chicken than in that of a young one. When the serum is heated at 56° and 70°C. for ½ hour, it becomes still more inhibiting. A substance favorable to cell activity has disappeared. It is therefore permissible to suppose that the growth-inhibiting power of serum and its variations are due to the antagonistic action of two substances, one growth-promoting and thermolabile, and the other growth-inhibiting and thermostable, the activating substance being always weaker in its effect than the inhibiting one. We know that activating substances can be extracted from embryonic tissue, from muscle and gland tissues, and from leucocytes of the adult animal, and that they are thermolabile and very unstable. Leucocytic secretions seem to have some of the properties of leucocytic extracts. It is probable that the activating substances which disappear from the heated serum are secreted by leucocytes and other cells. An increase of these secretions, then, would diminish the inhibiting action of serum on homologous fibroblasts. On the contrary, a decrease of the secretions in the serum would increase its inhibiting effect on homologous cells. The strong inhibiting action of serum in old age would be due partly to a reduction in the amount and activity of the substances secreted by leucocytes and tissue cells in the humors of the organism. Leucocytes also secreted in vitro substances which were toxic for foreign cells. Although the results were not constant, the serum appeared to become slightly more hemolytic for sheep or rabbit erythrocytes, under the influence of the leucocytes. The hemolysis of rabbit corpuscles by hen serum is due, according to Hyde,9 to a complex sensitizer alexin, and not merely to alexin, as Bordet thought. When a foreign protein was added to the culture medium, the leucocytic secretions increased, as was shown by the action on homologous fibroblasts of sera taken from cultures of leucocytes with and without casein. The presence in the medium of the cultures of leucocytes of only 0.1 per 1,000 casein did not markedly modify the action of their serum on the proliferation of fibroblasts. When the concentration of casein in the leucocyte cultures reached 1 per 1,000, the growth of chicken fibroblasts in the serum extracted from the Gabritschewski dishes became more rapid. But there was no parallel increase of the hemolytic action of the serum upon sheep erythrocytes. We found that chicken serum containing 0.1 per 1,000 casein was barely toxic for homologous fibroblasts, while it became markedly inhibiting when the casein concentration reached 1 per 1,000. Probably, there is a relation between the toxicity of the medium, the increase of leucocytic secretions, and the time of the increase. The change brought about by casein in the equilibrium of the system composed of the cells and their medium determines the secretion by the leucocytes of substances which increase the activity of homologous cells and oppose the inhibiting effect of the foreign proteins. This reaction of the leucocytes is immediate, and may represent the first defense of the organism against a factor which disturbs its equilibrium. Possibly it differs from the specific cell reaction which leads to the production of antibodies. It is known that antibodies develop more slowly. Hemolysins were detected in cultures of bone marrow 4 days after the addition of antigen. The immunization of fibroblasts against foreign proteins has been shown by Fischer to begin after 4 days. If leucocytes behave in the organism as they do in vitro, we may assume that before the appearance of antibodies, they respond to the presence of an antigen by setting free growth-promoting substances and possibly alexin. This immediate reaction of the leucocytes against a disturbing factor, and the resulting production of substances which increase the activity of homologous cells, might be partly responsible for the results observed in the treatment of certain diseases by the injection of foreign proteins. It may be concluded that, under the conditions of the experiments: 1. The serum obtained from cultures of leucocytes is less inhibiting for homologous fibroblasts than the serum from media without leucocytes. In some experiments, its hemolytic action on sheep or rabbit erythrocytes is also increased. 2. The addition of casein to leucocytic cultures brings about a decrease in the inhibiting effect of the serum on homologous fibroblasts. 3. The increase in the activity of homologous fibroblasts in serum obtained from leucocytic cultures is probably due to growth-promoting substances secreted by the leucocytes. The presence of a foreign protein under certain conditions determines a more abundant leucocytic secretion.
PMCID: PMC2128388  PMID: 19868699
14.  Performance of the PointCare NOW System for CD4 Counting in HIV Patients Based on Five Independent Evaluations 
PLoS ONE  2012;7(8):e41166.
Introduction
Point-of-care (POC) CD4 testing can improve access to treatment by enabling decentralization and reducing patient loss-to-follow-up. As new POC CD4 technologies become available, their performance should be assessed before widespread deployment. This study reports the findings of five independent evaluations of the PointCare NOW CD4 system.
Materials/Methods
Evaluations were conducted in Southern Africa (Mozambique, South Africa) and North America (Canada, USA). 492 blood samples (55 from HIV-negative blood donors and 437 from HIV-infected patients, including 20 children aged between 12 and 59 months) were tested with both the PointCare NOW and reference flow cytometry instruments. Assessment of bias, precision and levels of clinical misclassification for absolute and percent CD4 count was conducted.
Results
PointCare NOW significantly overestimated CD4 absolute counts with a mean relative bias of +35.0%. Bias was greater in samples with CD4 counts below ≤350cells/µl (+51.3%) than in the CD4 >350cells/µl stratum (15.1%). Bias in CD4% had a similar trend with an overall relative mean bias of +25.6% and a larger bias for low CD4 stratum (+40.2%) than the higher CD4 stratum (+5.8%). Relative bias for CD4% in children was −6.8%. In terms of repeatability, PointCare NOW had a coefficient of variation of 11%. Using a threshold of 350cells/µl, only 47% of patients who qualified for antiretroviral therapy with reference CD4 testing, would have been eligible for treatment with PointCare NOW test results. This was 39% using a 200cells/µl threshold. Agreement with infant samples was higher, with 90% qualifying at a 25% eligibility threshold.
Conclusion
The performance of the PointCare NOW instrument for absolute and percent CD4 enumeration was inadequate for HIV clinical management in adults. In children, the small sample size was not large enough to draw a conclusion. This study also highlights the importance of independent evaluation of new diagnostic technology platforms before deployment.
doi:10.1371/journal.pone.0041166
PMCID: PMC3415398  PMID: 22912668
15.  Mortality in Patients with HIV-1 Infection Starting Antiretroviral Therapy in South Africa, Europe, or North America: A Collaborative Analysis of Prospective Studies 
PLoS Medicine  2014;11(9):e1001718.
Analyzing survival in HIV treatment cohorts, Andrew Boulle and colleagues find mortality rates in South Africa comparable to or better than those in North America by 4 years after starting antiretroviral therapy.
Please see later in the article for the Editors' Summary
Background
High early mortality in patients with HIV-1 starting antiretroviral therapy (ART) in sub-Saharan Africa, compared to Europe and North America, is well documented. Longer-term comparisons between settings have been limited by poor ascertainment of mortality in high burden African settings. This study aimed to compare mortality up to four years on ART between South Africa, Europe, and North America.
Methods and Findings
Data from four South African cohorts in which patients lost to follow-up (LTF) could be linked to the national population register to determine vital status were combined with data from Europe and North America. Cumulative mortality, crude and adjusted (for characteristics at ART initiation) mortality rate ratios (relative to South Africa), and predicted mortality rates were described by region at 0–3, 3–6, 6–12, 12–24, and 24–48 months on ART for the period 2001–2010. Of the adults included (30,467 [South Africa], 29,727 [Europe], and 7,160 [North America]), 20,306 (67%), 9,961 (34%), and 824 (12%) were women. Patients began treatment with markedly more advanced disease in South Africa (median CD4 count 102, 213, and 172 cells/µl in South Africa, Europe, and North America, respectively). High early mortality after starting ART in South Africa occurred mainly in patients starting ART with CD4 count <50 cells/µl. Cumulative mortality at 4 years was 16.6%, 4.7%, and 15.3% in South Africa, Europe, and North America, respectively. Mortality was initially much lower in Europe and North America than South Africa, but the differences were reduced or reversed (North America) at longer durations on ART (adjusted rate ratios 0.46, 95% CI 0.37–0.58, and 1.62, 95% CI 1.27–2.05 between 24 and 48 months on ART comparing Europe and North America to South Africa). While bias due to under-ascertainment of mortality was minimised through death registry linkage, residual bias could still be present due to differing approaches to and frequency of linkage.
Conclusions
After accounting for under-ascertainment of mortality, with increasing duration on ART, the mortality rate on HIV treatment in South Africa declines to levels comparable to or below those described in participating North American cohorts, while substantially narrowing the differential with the European cohorts.
Please see later in the article for the Editors' Summary
Editors' Summary
Background
AIDS has killed about 36 million people since the first recorded case of the disease in 1981, and a similar number of people (including 25 million living in sub-Saharan Africa) are currently infected with HIV, the virus that causes AIDS. HIV destroys immune system cells (including CD4 cells, a type of lymphocyte), leaving infected individuals susceptible to other serious infections. Early in the AIDS epidemic, HIV-positive people usually died within 10 years of becoming infected. In 1996, effective antiretroviral therapy (ART) became available and, for people living in high-income countries, HIV infection became a chronic condition. But ART was expensive, so HIV/AIDS remained largely untreated and fatal in resource-limited countries. Then, in 2003, the international community began to work towards achieving universal access to ART. By the end of 2012, nearly two-thirds of HIV-positive people (nearly 10 million individuals) living in low- and middle-income countries who were eligible for treatment because their CD4 cell count had fallen below 350/mm3 blood or because they had developed an AIDS-defining condition were receiving treatment.
Why Was This Study Done?
It is known that a larger proportion of HIV-positive patients starting ART die during the first year of treatment in sub-Saharan Africa than in Europe and North America. This difference arises in part because patients in resource-limited settings tend to have lower CD4 counts when they start treatment than patients in wealthy countries. However, the lack of reliable data on mortality (death) in resource-limited settings has made it hard to compare longer-term outcomes in different settings. Information on the long-term outcomes of HIV-positive patients receiving ART in resource-limited countries is needed to guide the development of appropriate health systems and treatment regimens in these settings. In this collaborative analysis of prospective cohort studies, the researchers compare mortality up to 4 years on ART in South Africa, Europe, and North America. A prospective cohort study follows a group of individuals over time to see whether differences in specific characteristics at the start of the study affect subsequent outcomes. A collaborative analysis combines individual patient data from several studies.
What Did the Researchers Do and Find?
The researchers combined data from four South Africa cohorts of HIV-positive patients starting ART included in the International Epidemiologic Databases to Evaluate AIDS South African (IeDEA-SA) collaboration with data from six North American cohorts and nine European cohorts included in the ART Cohort Collaboration (ART-CC). The South African cohorts were chosen because unusually for studies undertaken in countries in sub-Saharan Africa the vital status of patients (whether they had died) who had been lost to follow-up in these cohorts could be obtained from the national population register. Patients in South Africa began treatment with more advanced disease (indicated by a lower average CD4 count) than patients in Europe or North America. Notably, high early mortality after starting ART in South Africa occurred mainly in patients starting ART with a CD4 count below 50 cells/mm3. The cumulative mortality after 4 years of ART was 16.6%, 4.7%, and 15.3% in South Africa, Europe, and North America, respectively. After adjusting for patient characteristics at ART initiation, the mortality rate among patients beginning ART was initially lower in Europe and North American than in South Africa. However, although the adjusted mortality rate in Europe remained lower than the rate in South Africa, the rate in North America was higher than that in South Africa between 24 and 48 months on ART.
What Do These Findings Mean?
Although the linkage to national vital registration systems (databases of births and deaths) undertaken in this collaborative analysis is likely to have greatly reduced bias due to under-ascertainment of mortality, the accuracy of these findings may still be limited by differences in how this linkage was undertaken in different settings. Nevertheless, these findings suggest that mortality among HIV-infected patients receiving ART in South Africa, although initially higher than in Europe and North America, rapidly declines with increasing duration on ART and, after 4 years of treatment, approaches the rate seen in high-income settings. Intriguingly, these findings also highlight the relatively higher late mortality in North America compared to either Europe or South Africa, a result that needs to be investigated to explore the extent to which differences in mortality ascertainment, patient characteristics and comorbidities, or health systems and treatment regimens contribute to variations in outcomes among HIV-positive patients in various settings.
Additional Information
Please access these websites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.1001718.
This study is further discussed in a PLOS Medicine Perspective by Agnes Binagwaho and colleagues
Information is available from the US National Institute of Allergy and Infectious Diseases on HIV infection and AIDS
NAM/aidsmap provides basic information about HIV/AIDS, and summaries of recent research findings on HIV care and treatment
Information is available from Avert, an international AIDS charity, on many aspects of HIV/AIDS, including information on universal access to ART, on HIV and AIDS in sub-Saharan Africa, and on HIV and AIDS in South Africa (in English and Spanish)
The World Health Organization provides information on all aspects of HIV/AIDS (in several languages); its 2013 Consolidated guidelines on the use of antiretroviral drugs for treating and preventing HIV infections: recommendations for a public health approach are available
The 2013 UNAIDS World AIDS Day Report provides up-to-date information about the AIDS epidemic and efforts to halt it
Information about the International Epidemiologic Databases to Evaluate AIDS South African (IeDEA-SA) collaboration and about the ART Cohort Collaboration is available
Personal stories about living with HIV/AIDS are available through Avert, Nam/aidsmap, and Healthtalkonline
doi:10.1371/journal.pmed.1001718
PMCID: PMC4159124  PMID: 25203931
16.  Single-platform, volumetric, CD45-assisted pan-leucogating flow cytometry for CD4 T lymphocytes monitoring of HIV infection according to the WHO recommendations for resource-constrained settings 
BMC Research Notes  2013;6:169.
Background
Validation of new affordable CD4 T cell measurement technologies is crucial specifically in resource-poor countries for antiretroviral treatment eligibility and immunologic CD4 monitoring of HIV-infected patients.
Methods
The absolute and percentage CD4 T cell counts of 258 HIV-1-infected blood samples (182 adults and 76 children), living in N’Djamena, Chad, were performed by single-platform, volumetric, CD45-assisted pan-leucogating Auto40 flow cytometer (Apogee Flow Systems Ltd, Hemel Hempstead, UK) comparing to the FACSCalibur flow cytometer as a reference method.
Results
Absolute and percentage CD4 T cell counts obtained by Auto40 and FACSCalibur of 258 HIV-1-infected blood samples were highly correlated (r = 0.99 and r = 0.96, respectively). The mean absolute bias and percent bias between Apogee Auto40 and FACSCalibur absolute CD4 T cell counts, were −9.4 cells/μl with limits of agreement from −15 to 93 cells/μl, and +2.0% with limits of agreement from −0.9 to 4.9%, respectively. The mean of absolute bias and percent bias between Apogee Auto40 and FACSCalibur of CD4 percentage results were +0.4% (95% CI: -0.02 – 0.86) with limits of agreement from −2.4 to 0.3%, and +3.0% with limits of agreement from −6.6 to 0.6%, respectively. The Auto40 counting allowed to identify the majority of adults with CD4 T cells below 200 cells/μl (sensitivity: 89%; specificity: 99%) or below 350 cells/μl (sensitivity: 94%; specificity:98%); and of children below 750 cells/μl (sensitivity: 99%; specificity: 96%) or below 25% CD4+ (sensitivity: 94%; specificity: 98%).
Conclusion
The Auto40 analyzer is an alternative flow cytometer for CD4 T lymphocyte enumeration to be used in routine for immunological monitoring according to the current WHO recommendations in HIV-infected adults as well as children living in resource-constrained settings like Chad.
doi:10.1186/1756-0500-6-169
PMCID: PMC3653683  PMID: 23631664
Flow cytometry; CD4 T cell count; Pan-leucogating; CD45; Sub-Saharan Africa; Chad
17.  HIV-1 Transmission during Early Infection in Men Who Have Sex with Men: A Phylodynamic Analysis 
PLoS Medicine  2013;10(12):e1001568.
Erik Volz and colleagues use HIV genetic information from a cohort of men who have sex with men in Detroit, USA to dissect the timing of onward transmission during HIV infection.
Please see later in the article for the Editors' Summary
Background
Conventional epidemiological surveillance of infectious diseases is focused on characterization of incident infections and estimation of the number of prevalent infections. Advances in methods for the analysis of the population-level genetic variation of viruses can potentially provide information about donors, not just recipients, of infection. Genetic sequences from many viruses are increasingly abundant, especially HIV, which is routinely sequenced for surveillance of drug resistance mutations. We conducted a phylodynamic analysis of HIV genetic sequence data and surveillance data from a US population of men who have sex with men (MSM) and estimated incidence and transmission rates by stage of infection.
Methods and Findings
We analyzed 662 HIV-1 subtype B sequences collected between October 14, 2004, and February 24, 2012, from MSM in the Detroit metropolitan area, Michigan. These sequences were cross-referenced with a database of 30,200 patients diagnosed with HIV infection in the state of Michigan, which includes clinical information that is informative about the recency of infection at the time of diagnosis. These data were analyzed using recently developed population genetic methods that have enabled the estimation of transmission rates from the population-level genetic diversity of the virus. We found that genetic data are highly informative about HIV donors in ways that standard surveillance data are not. Genetic data are especially informative about the stage of infection of donors at the point of transmission. We estimate that 44.7% (95% CI, 42.2%–46.4%) of transmissions occur during the first year of infection.
Conclusions
In this study, almost half of transmissions occurred within the first year of HIV infection in MSM. Our conclusions may be sensitive to un-modeled intra-host evolutionary dynamics, un-modeled sexual risk behavior, and uncertainty in the stage of infected hosts at the time of sampling. The intensity of transmission during early infection may have significance for public health interventions based on early treatment of newly diagnosed individuals.
Please see later in the article for the Editors' Summary
Editors' Summary
Background
Since the first recorded case of AIDS in 1981, the number of people infected with HIV, the virus that causes AIDS, has risen steadily. About 34 million people are currently HIV-positive, and about 2.5 million people become newly infected with HIV every year. Because HIV is usually transmitted through unprotected sex with an infected partner, individuals can reduce their risk of infection by abstaining from sex, by having only one or a few partners, and by always using condoms. Most people do not become ill immediately after infection with HIV, although some develop a short flu-like illness. The next stage of HIV infection, which may last more than ten years, also has no major symptoms, but during this stage, HIV slowly destroys immune system cells. Eventually, the immune system can no longer fight off infections by other disease-causing organisms, and HIV-positive people then develop one or more life-threatening AIDS-defining conditions, including unusual infections and specific types of cancer. HIV infection can be controlled, but not cured, by taking a daily cocktail of antiretroviral drugs.
Why Was This Study Done?
The design of effective programs to prevent the spread of HIV/AIDS depends on knowing how HIV transmissibility varies over the course of HIV infection. Consider, for example, a prevention strategy that focuses on increasing treatment rates: antiretroviral drugs, in addition to reducing illness and death among HIV-positive people, reduce HIV transmission from HIV-positive individuals. “Treatment as prevention” can only block transmissions that occur after diagnosis and entry into care. However, the transmissibility of HIV per sexual contact depends on a person's viral load, which peaks during early HIV infection, when people are often unaware of their HIV status and may still be following the high-risk patterns of sexual behavior that caused their own infection. Epidemiological surveillance data (information on HIV infections within populations) can be used to estimate how many new HIV infections occur within a population annually (HIV incidence) and the proportion of the population that is HIV-positive (HIV prevalence), but cannot be used to estimate the timing of transmission events. In this study, the researchers use “phylodynamic analysis” to estimate HIV incidence and prevalence and the timing of HIV transmission during infection. HIV, like many other viruses, rapidly accumulates genetic changes. The timing of transmission influences the pattern of these changes. Viral phylodynamic analysis—the quantitative study of how epidemiological, immunological, and evolutionary processes shape viral phylogenies (evolutionary trees)—can therefore provide estimates of transmission dynamics.
What Did the Researchers Do and Find?
The researchers obtained HIV sequence data (collected for routine surveillance of antiretroviral resistance mutations) and epidemiological surveillance data (including information on the stage of infection at diagnosis) for 662 HIV-positive men who have sex with men living in the Detroit metropolitan area of Michigan. They constructed a phylogenetic tree from the sequences using a “relaxed clock” approach and then fitted an epidemiological model (a mathematical model that represents the progress of individual patients through various stages of HIV infection) to the sequence data. Their approach, which integrates surveillance data and genetic data, yielded estimates of HIV incidence and prevalence among the study population similar to those obtained from surveillance data alone. However, it also provided information about HIV transmission that could not be obtained from surveillance data alone. In particular, it allowed the researchers to estimate that, in the current HIV epidemic among men who have sex with men in Detroit, 44.7% of HIV transmissions occur during the first year of infection.
What Do These Findings Mean?
The robustness of these findings depends on the validity of the assumptions included in the researchers' population genetic model and on the accuracy of the data fed into the model, and may not be generalizable to other cities or to other risk groups. Nevertheless, the findings of this analysis, which can be repeated in any setting where HIV sequence data for individual patients can be linked to patient-specific clinical and behavioral information, have important implications for HIV control strategies based on the early treatment of newly diagnosed individuals. Because relatively few infected individuals are diagnosed during early HIV infection, when the HIV transmission rate is high, it is unlikely, suggest the researchers, that the “treatment as prevention” strategy will effectively control the spread of HIV unless there are very high rates of HIV testing and treatment.
Additional Information
Please access these websites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.1001568.
This study is further discussed in a PLOS Medicine Perspective by Timothy Hallett
Information is available from the US National Institute of Allergy and Infectious Diseases on HIV infection and AIDS
NAM/aidsmap provides basic information about HIV/AIDS and summaries of recent research findings on HIV care and treatment
Information is available from Avert, an international AIDS charity, on many aspects of HIV/AIDS, including information on HIV treatment as prevention (in English and Spanish)
The PLOS Medicine Collection Investigating the Impact of Treatment on New HIV Infections provides more information about HIV treatment as prevention
A PLOS Computational Biology Topic Page (a review article that is a published copy of record of a dynamic version of the article as found in Wikipedia) about viral phylodynamics is available
The US National Institute of Health–funded HIV Sequence Database contains HIV sequences and tools to analyze these sequences
Patient stories about living with HIV/AIDS are available through Avert; the charity website Healthtalkonline also provides personal stories about living with HIV
doi:10.1371/journal.pmed.1001568
PMCID: PMC3858227  PMID: 24339751
18.  Antigen Load and Viral Sequence Diversification Determine the Functional Profile of HIV-1–Specific CD8+ T Cells 
PLoS Medicine  2008;5(5):e100.
Background
Virus-specific CD8+ T lymphocytes play a key role in the initial reduction of peak viremia during acute viral infections, but display signs of increasing dysfunction and exhaustion under conditions of chronic antigen persistence. It has been suggested that virus-specific CD8+ T cells with a “polyfunctional” profile, defined by the capacity to secrete multiple cytokines or chemokines, are most competent in controlling viral replication in chronic HIV-1 infection. We used HIV-1 infection as a model of chronic persistent viral infection to investigate the process of exhaustion and dysfunction of virus-specific CD8+ T cell responses on the single-epitope level over time, starting in primary HIV-1 infection.
Methods and Findings
We longitudinally analyzed the polyfunctional epitope-specific CD8+ T cell responses of 18 patients during primary HIV-1 infection before and after therapy initiation or sequence variation in the targeted epitope. Epitope-specific CD8+ T cells responded with multiple effector functions to antigenic stimulation during primary HIV-1 infection, but lost their polyfunctional capacity in response to antigen and up-regulated programmed death 1 (PD-1) expression with persistent viremic infection. This exhausted phenotype significantly decreased upon removal of stimulation by antigen, either in response to antiretroviral therapy or by reduction of epitope-specific antigen load in the presence of ongoing viral replication, as a consequence of in vivo selection of cytotoxic T lymphocyte escape mutations in the respective epitopes. Monofunctionality increased in CD8+ T cell responses directed against conserved epitopes from 49% (95% confidence interval 27%–72%) to 76% (56%–95%) (standard deviation [SD] of the effect size 0.71), while monofunctionality remained stable or slightly decreased for responses directed against escaped epitopes from 61% (47%–75%) to 56% (42%–70%) (SD of the effect size 0.18) (p < 0.05).
Conclusion
These data suggest that persistence of antigen can be the cause, rather than the consequence, of the functional impairment of virus-specific T cell responses observed during chronic HIV-1 infection, and underscore the importance of evaluating autologous viral sequences in studies aimed at investigating the relationship between virus-specific immunity and associated pathogenesis.
Marcus Altfeld and colleagues suggest that the exhaustion of virus-specific CD8+ T cells during chronic HIV infection likely results from the persistence of antigen.
Editors' Summary
Background.
Viruses are small infectious agents responsible for many human diseases, including acquired immunodeficiency syndrome (AIDS). Like other viruses, the human immunodeficiency virus 1 (HIV-1; the cause of AIDS) enters human cells and uses the cellular machinery to replicate before bursting out of its temporary home. During the initial stage of HIV infection, a particular group of cells in the human immune system, CD8+ T cells, are thought to be important in controlling the level of the virus. These immune system cells recognize pieces of viral protein called antigens displayed on the surface of infected cells; different subsets of CD8+ T cells recognize different antigens. When a CD8+ T cell recognizes its specific antigen (or more accurately, a small part of the antigen called an “epitope”), it releases cytotoxins (which kill the infected cells) and cytokines, proteins that stimulate CD8+ T cell proliferation and activate other parts of the immune system. With many viruses, when a person first becomes infected (an acute viral infection), antigen-specific CD8+ T cells completely clear the infection. But with HIV-1 and some other viruses, these cells do not manage to remove all the viruses from the body and a chronic (long-term) infection develops, during which the immune system is constantly exposed to viral antigen.
Why Was This Study Done?
In HIV-1 infections (and other chronic viral infections), virus-specific CD8+ T cells lose their ability to proliferate, to make cytokines, and to kill infected cells as patients progress to the long-term stages of infection. That is, the virus-specific CD8+ T cells gradually lose their “effector” functions and become functionally impaired or “exhausted.” “Polyfunctional” CD8+ T cells (those that release multiple cytokines in response to antigen) are believed to be essential for an effective CD8+ T cell response, so scientists trying to develop HIV-1 vaccines would like to stimulate the production of this type of cell. To do this they need to understand why these polyfunctional cells are lost during chronic infections. Is their loss the cause or the result of viral persistence? In other words, does the constant presence of viral antigen lead to the exhaustion of CD8+ T cells during chronic HIV infection? In this study, the researchers investigate this question by looking at the polyfunctionality of CD8+ cells responding to several different viral epitopes at various times during HIV-1 infection, starting very early after infection with HIV-1 had occurred.
What Did the Researchers Do and Find?
The researchers enrolled 18 patients recently infected with HIV-1 and analyzed their CD8+ T cell responses to specific epitopes at various times after enrollment using a technique called flow cytometry. They found that the epitope-specific CD8+ cells produced several effector proteins after antigen stimulation during the initial stage of HIV-1 infection, but lost their polyfunctionality in the face of persistent viral infection. The CD8+ T cells also increased their production of programmed death 1 (PD-1), a protein that has been shown to be associated with the functional impairment of CD8+ T cells. Some of the patients began antiretroviral therapy during the study, and the researchers found that this treatment, which reduced the viral load, reversed CD8+ T cell exhaustion. Finally, the appearance in the patients' blood of viruses that had made changes in the specific epitopes recognized by the CD8+ T cells to avoid being killed by these cells, also reversed the exhaustion of the T cells recognizing these particular epitopes.
What Do These Findings Mean?
These findings suggest that the constant presence of HIV-1 antigen causes the functional impairment of virus-specific CD8+ T cell responses during chronic HIV-1 infections. Treatment with antiretroviral drugs reversed this functional impairment by reducing the amount of antigen in the patients. Similarly, the appearance of viruses with altered epitopes, which effectively reduced the amount of antigen recognized by those epitope-specific CD8+ T cells without reducing the viral load, also reversed T cell exhaustion. These results would not have been seen if the functional impairment of CD8+ cells were the cause rather than the result of antigen persistence. By providing new insights into how the T cell response to viruses evolves during persistent viral infections, these findings should help in the design of vaccines against HIV and other viruses that cause chronic viral infections.
Additional Information.
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0050100.
Read a related PLoS Medicine Research in Translation article
Learn more from the researchers' Web site, the Partners AIDS Research Center
Wikipedia has a page on cytotoxic T cells (note that Wikipedia is a free online encyclopedia that anyone can edit; available in several languages)
Information is available from the US National Institute of Allergy and Infectious Diseases on HIV infection and AIDS
HIV InSite has comprehensive information on all aspects of HIV/AIDS, including a detailed article on the immunopathogenesis of HIV infection
NAM, a UK registered charity, provides information about all aspects of HIV and AIDS, including a fact sheet on the stages of HIV infection and on the immune response to HIV
Information is available from Avert, an international AIDS charity, on all aspects of HIV/AIDS, including information on the stages of HIV infection
doi:10.1371/journal.pmed.0050100
PMCID: PMC2365971  PMID: 18462013
19.  Expanding Access to HIV Viral Load Testing: A Systematic Review of RNA Stability in EDTA Tubes and PPT beyond Current Time and Temperature Thresholds 
PLoS ONE  2014;9(12):e113813.
Background
HIV viral load (VL) testing is the gold standard for antiretroviral treatment monitoring, but many barriers exist to VL testing in resource-limited settings, including storage and transport limitations for whole blood and plasma. Data from various studies indicate that HIV RNA is stable beyond current recommendations. We conducted a systematic review to assess stability data of HIV RNA in whole blood and plasma across times and temperatures.
Methods and Findings
Using a pre-defined protocol, five databases were searched for studies where blood samples from HIV patients were stored at time and temperature points that exceeded manufacturer recommendations. RNA stability, the primary outcome, was measured by the difference in means compared to samples stored within established thresholds. RNA stability was defined as ≤0.5 log degradation. The search identified 10,716 titles, of which nine full-text articles were included for review. HIV RNA maintained stability in EDTA whole blood and plasma at all measured time points up to 168 hours when stored at 4°C, while stability was detected at 72 hours (95% confidence) in whole blood at 25°C, with data points before and beyond 72 hours suggesting stability but not reaching statistical significance. For EDTA plasma stored at 30°C, stability was maintained up to 48 hours (95% confidence), with OLS linear regression estimates up to 127 hours, suggesting stability. Overall, quality of studies was moderate. Limitations included small sample sizes, few studies meeting inclusion criteria, and no studies examining RNA stability in low viremia (<3,000 copies/mL) environments.
Conclusions
Whole blood and plasma samples in EDTA may remain stable under conditions exceeding current manufacturer recommendations for HIV VL testing. However, given the limited number of studies addressing this question, especially at low levels of viremia, additional evaluations on HIV RNA stability in EDTA tubes and PPT in field conditions are needed.
doi:10.1371/journal.pone.0113813
PMCID: PMC4249975  PMID: 25437009
20.  The Leukocyte Culture Method in the Diagnosis of Free-martinism 
The clinical application and reliability of the leukocyte culture method for the diagnosis of freemartinism were examined and the length of time that blood samples could be held at room temperature and in the refrigerator prior to culturing, was investigated.
The chromosome findings by the leukocyte culture method in 14 freemartins and 9 non-freemartin females belonging to heterosexual twins or triplets revealed that XX-XY cell chimerism exists only in the former, whereas the latter were exclusively of normal female complement.
The mitotic index in bovine blood after preservation for varying periods was studied on samples from two animals. Blood samples from these two animals stored at 5°C for 6 hours in a refrigerator showed the mitotic index to be 3.8 and 5.3 per cent which gradually decreased in samples stored for longer than 12 hours. After 72 hours, a very rapid decrease in mitotic index occurred in both cases, reaching zero in samples stored for 96 and 108 hours. Samples kept at room temperature followed a similar pattern as under refrigeration but with slightly lower values throughout.
Images
PMCID: PMC1319303  PMID: 4234791
21.  Stability of Sodium Nitroprusside and Sodium Thiosulfate 1:10 Intravenous Admixture 
Hospital pharmacy  2010;45(10):779-784.
Purpose
Thiosulfate has been shown to reduce the risk of cyanide toxicity during nitroprusside administration. Admixtures containing both agents may provide a safe and effective alternative to more expensive agents used to reduce blood pressure in the critically ill patient. This study determined the physical and chemical stability of a 1:10 nitroprusside:thiosulfate admixture, stored up to 48 hours. The economic consequences of a shift toward using thiosulfate and nitroprusside, and away from higher cost alternatives, are considered.
Methods
Seven samples of 50 mg nitroprusside and 500 mg thiosulfate were prepared and stored away from light, at room temperature, and in a refrigerator prepared in D5W and NS. Each sample was analyzed via a novel high-performance liquid chromatographic (HPLC) method at time 0, 8, 24, and 48 hours. The method was tested and passed specifications for linearity, reproducibility, and accuracy. A visual inspection by 9 licensed pharmacists was used to demonstrate physical stability. A cost evaluation comparing nitroprusside and thiosulfate to alternative agents was completed.
Results
The concentration of both nitroprusside and thiosulfate remain greater than 95% of the initial concentration through 48 hours. Physical compatibility was confirmed in all samples tested through 72 hours.
Conclusion
The combination of nitroprusside and thiosulfate is chemically and physically stable as a single compounded dose for up to 48 hours when stored at room temperature and protected from light. The admixture represents an inexpensive option to other higher cost alternatives such as nicardipine or clevidipine.
doi:10.1310/hpj4510-779
PMCID: PMC3102563  PMID: 21625332
drug stability; high-pressure liquid chromatography; pharmacoeconomics
22.  Estimating Incidence from Prevalence in Generalised HIV Epidemics: Methods and Validation 
PLoS Medicine  2008;5(4):e80.
Background
HIV surveillance of generalised epidemics in Africa primarily relies on prevalence at antenatal clinics, but estimates of incidence in the general population would be more useful. Repeated cross-sectional measures of HIV prevalence are now becoming available for general populations in many countries, and we aim to develop and validate methods that use these data to estimate HIV incidence.
Methods and Findings
Two methods were developed that decompose observed changes in prevalence between two serosurveys into the contributions of new infections and mortality. Method 1 uses cohort mortality rates, and method 2 uses information on survival after infection. The performance of these two methods was assessed using simulated data from a mathematical model and actual data from three community-based cohort studies in Africa. Comparison with simulated data indicated that these methods can accurately estimates incidence rates and changes in incidence in a variety of epidemic conditions. Method 1 is simple to implement but relies on locally appropriate mortality data, whilst method 2 can make use of the same survival distribution in a wide range of scenarios. The estimates from both methods are within the 95% confidence intervals of almost all actual measurements of HIV incidence in adults and young people, and the patterns of incidence over age are correctly captured.
Conclusions
It is possible to estimate incidence from cross-sectional prevalence data with sufficient accuracy to monitor the HIV epidemic. Although these methods will theoretically work in any context, we have able to test them only in southern and eastern Africa, where HIV epidemics are mature and generalised. The choice of method will depend on the local availability of HIV mortality data.
Timothy Hallett and colleagues develop and test two user-friendly methods to estimate HIV incidence based on changes in cross-sectional prevalence, using either mortality rates or survival after infection.
Editors' Summary
Background.
More than 25 million people have died from AIDS and about 33 million people are currently infected with human immunodeficiency virus (HIV, the virus that causes AIDS). Faced with this threat to human health, governments and international agencies are working together to halt the AIDS epidemic. An important part of this effort is HIV surveillance. The spread of HIV needs to be monitored to assess the impact of interventions (for example, the provision of antiretroviral drugs) and to plan for current and future health care needs. HIV surveillance in countries where the epidemic has spread beyond specific groups into the whole population (a generalized epidemic) has mainly relied on determining the prevalence of HIV infection (the fraction of the population that is infected) among women attending antenatal clinics. Recently, however, household health surveys (for example, the Demographic and Health Surveys) have begun to use blood testing for antibodies to the AIDS virus (serological testing) to provide more accurate estimates of HIV prevalence in the general adult population.
Why Was This Study Done?
Although prevalence estimates provide useful information about the HIV epidemic, another important indicator is incidence—the number of new infections occurring during a specific time period. Incidence measurements provide more information about temporal changes in the epidemic and transmission patterns and allow public-health experts to make better predictions of future health care needs. But, whereas prevalence can be measured with anonymized serological surveys, individuals would have to be identified and followed up in repeat serological surveys to provide a direct measurement of incidence. This is expensive and hard to achieve in many settings. In this study, therefore, the researchers develop and validate two mathematical methods to estimate HIV incidence in generalized HIV epidemics from prevalence data.
What Did the Researchers Do and Find?
Changes in the fraction of the population living with HIV (prevalence) can occur not only because of changes in the rate of new infections (incidence), but also because mortality rates are much higher for infected individuals than others. The researchers' methods disentangle the contributions to HIV prevalence (as measured in serological surveys) made by new infections from those due to deaths from AIDS and other causes. Their first method incorporates information on death rates collected in cohort studies of HIV infection (cohort studies investigate outcomes in groups of people); their second method uses information on survival after HIV infection, also collected in long-running cohort studies. The accuracy of both methods was assessed using computer-simulated data and actual data on HIV prevalence and incidence collected in three community-based cohort studies in Zimbabwe and Uganda (countries with generalized but declining HIV epidemics) and Tanzania (a country with a generalized, stable epidemic). Both methods provided accurate estimates of HIV incidence from the simulated data. Using the data collected in Africa, the mean difference between actual measurements of incidence and the estimate provided by method 1 was 19%; for method 2 it was 14%. In addition, the measured and estimated incidences were in good agreement for all age groups.
What Do These Findings Mean?
These findings suggest HIV incidence rates can be estimated from repeat surveys of prevalence with sufficient accuracy to monitor the HIV epidemic. The accuracy of the estimates across all age groups is particularly important because knowledge of the age-related risk pattern provides the information on transmission patterns that is needed to design effective intervention programs. Because these methods were tested using data only from southern and eastern Africa where the HIV epidemic is mature and generalized, they may not work as well in regions where the epidemic is restricted to subsets of the population. Other factors that might affect their accuracy include the amount of international migration and the uptake of antiretroviral therapies. Nevertheless, with the increased availability of serial measurements of serological prevalence, these new methods for estimating HIV incidence from HIV prevalence could prove extremely useful for monitoring the progress of national HIV epidemics and for guiding HIV control programs. The authors include spreadsheets that can be used to calculate incidence by either method from consecutive survey data.
Additional Information.
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0050080.
The US National Institute of Allergy and Infectious Diseases provides information on HIV infection and AIDS
The US Centers for Disease Control and Prevention provides information on global HIV/AIDS topics (in English and Spanish)
The HIV InSite provides comprehensive and up-to-date information on all aspects of HIV/AIDS from the University of California San Francisco, including country reports on the AIDS epidemic in 195 countries, including Uganda, Zimbabwe, and Tanzania
Avert, an international AIDS charity, provides information on all aspects of HIV/AIDS, including fact sheets on understanding HIV and AIDS statistics, and on HIV and AIDS in Africa
The Demographic and Health Surveys program collects, analyzes, and disseminates information on health and population trends in countries around the world
doi:10.1371/journal.pmed.0050080
PMCID: PMC2288620  PMID: 18590346
23.  Factors affecting preconditioning of Trichinella spiralis nativa larvae in musculature to low temperatures. 
The influence of preconditioning temperature, length of the preconditioning period, host and age of the infection on the survival of Trichinella spiralis nativa larvae in musculature to low temperature refrigeration was investigated. Dogs, foxes, ferrets, mink and guinea pigs were infected with a T. spiralis nativa isolate, killed at various times postinfection, preconditioned at temperature of -10 degrees C, -15 degrees C or -20 degrees C for varying periods of time prior to low temperature refrigeration and subsequent pepsin digestion to determine survival of larvae. The preconditioning temperature played an important role in the subsequent survival of larvae in musculature at low refrigeration temperatures. Under the conditions of this study, survival of larvae was greater as the preconditioning temperature became lower. The minimum period of preconditioning required had an inverse relationship with the refrigeration temperature. Preconditioning of the T. spiralis nativa isolate used occurred in the musculature of guinea pigs, foxes, ferrets, mink and dogs with larvae surviving longer in vulpine and canine musculature than in the other hosts studied. Age of the infection was not a major factor in the survival of preconditioned larvae in musculature at low refrigeration temperatures although survival was slightly longer in older infections.
PMCID: PMC1255297  PMID: 3607648
24.  Effect of a Common Diet and Regular Beverage on Enamel Erosion in Various Temperatures: An In-Vitro Study 
Objective:
This study compared diet and regular Coca-Cola on enamel erosion in cold and room temperatures.
Materials and Methods:
Seventy five enamel specimens were prepared and divided into 5 equal groups (N=15) as follows: Group 1: regular beverage at room temperature, Group 2: regular beverage at refri-gerator temperature, Group 3: diet beverage at room and Group 4: diet beverage at refrige-rator temperature. The specimens were immersed in the regular or diet beverage (Coca-Cola, trade mark regd. Khoshgovar Co., Tehran, Iran) at room (20°C) or refrigerator (2°C) temperatures for 20 minutes, 3 times per day for 7 days. Specimens in the control subjects (group 5) were placed in synthetic saliva at room temperature for 7 days. The hardness of specimens was tested using Vickers test under 500 gr loads for 5 seconds. The data were analyzed using two-way ANOVA and Tukey tests.
Results:
The mean and standard deviations of micro-hardness values of the studied groups were as follow: G1: 304.26±29.71, G2: 285.53±42.14, G3: 279.06±39.52, G4: 266.80±23.98 and G5: 319± 30.79. There was a significant difference in the beverage type as the main factor (p<0.05), but temperature factor and their interaction effect on enamel hardness showed no significant difference (p>0.05). Tukey tests showed that there were significant differences between control and diet groups as well as regular and diet groups.
Conclusion:
Diet Coca-Cola is more erosive than the regular type and the temperature of the beverages used had no significant influence on enamel erosion.
PMCID: PMC4025425  PMID: 24910648
Beverages; Cola; Tooth Erosions; Enamel
25.  Stability of Epinephrine at Standard Concentrations 
Background
To minimize medication errors, standard concentrations are recommended for medications intended for continuous infusion in pediatric patients. Premixing of epinephrine (commonly used to manage septic shock in children) would improve timeliness, safety, and cost-effectiveness. However, information about the stability of epinephrine at standard concentrations is limited.
Objectives:
To evaluate the stability of epinephrine in 5% dextrose in water at standard concentrations and to extend its expiration date after storage in infusion bags at 4°C and 25°C for up to 30 days.
Methods:
A total of 6 infusion bags were prepared with 200 mL of epinephrine solution, 2 bags for each of 3 standard concentrations (25, 50, and 100 μg/mL). Three bags (one for each concentration) were stored under refrigeration (4°C), and the remaining 3 bags were stored at room temperature (25°C). Physical characteristics (including pH, colour, and presence of precipitate) were evaluated daily for the first 14 days and every 1 to 5 days thereafter until day 30. Three 1.5-mL samples were collected from each bag immediately after preparation (time 0), every 24 h (at 24 h, 48 h, 72 h, 96 h, etc.) for the first 14 days, and every 1 to 5 days thereafter until day 30. Each sample was analyzed by stability-indicating high-performance liquid chromatography. A solution was considered stable if it maintained at least 90% of its initial concentration.
Results:
No notable changes in pH, colour, or precipitation were observed in any of the solutions after storage at 4°C or 25°C for up to 30 days. All formulations maintained more than 95% of the initial epinephrine concentration on day 30. In addition, the calculated lower limit of the 95% confidence interval indicated that 93% or more of the initial concentration remained on day 30.
Conclusions:
Preparations of epinephrine were stable for up to 30 days, with or without refrigeration. Because stability alone does not guarantee bioavailability or efficacy of a drug, future clinical studies are recommended to evaluate the pharmacokinetics and pharmacodynamics of these formulations.
PMCID: PMC4071081  PMID: 24970939
epinephrine; standard concentration; stability; épinéphrine; concentration standard; stabilité

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