Viewpoint: Why do we need a point-of-care CD4 test for low-income countries?

In this paper, we discuss the reasons why we urgently need a point-of-care (POC) CD4 test, elaborate the problems we have experienced with the current technology which hampers CD4-count coverage and highlight the ideal characteristics of a universal CD4 POC test. It is high-time that CD4 technology is simplified and adapted for wider use in low-income countries to change the current paradigm of restricted access once and for all.

Dried blood spots are a useful tool for quality assurance of rapid HIV testing in Kigali, Rwanda.

A study was conducted in two primary health facilities in Kigali, Rwanda, to determine whether dried blood spots (DBS) used for quality control of HIV testing would give comparable results with serum after being stored for a period of 14 days and 30 days at ambient temperature. DBS and serum specimens were collected from patients undergoing HIV testing. ELISA performed on serum at baseline (gold standard) was compared with DBS results. The study included a total of 491 patients, comprising 92 (19%) males and 399 (81%) females with a median age of 27 years. A total of 148 individuals (30%) were HIV-positive. The average ambient temperature under which DBS specimens were stored at the health facilities was 23 degrees C (range 18-25 degrees C). The kappa statistic at 14 days and 30 days was 0.99 (99.4% agreement) and 0.98 (99.2% agreement), respectively, signifying almost 'perfect agreement (P<0.001)' with the gold standard. In a resource-limited sub-Saharan African country embarking on scaling-up of HIV testing, DBS stored at ambient conditions for up to 1 month were found to be a useful and robust tool to perform quality control of rapid HIV testing at the health centre level.

The Partec CyFlow Counter could provide an option for CD4+ T-cell monitoring in the context of scaling-up antiretroviral treatment at the district level in Malawi.

A study was conducted in rural Malawi to verify (a) whether the Partec CyFlow Counter((R)) for CD4+ T-cell lymphocyte counting in HIV-positive individuals could be introduced into a district hospital laboratory and (b) whether it would produce CD4 counts of acceptable quality. CD4+ cell counting was performed using the Partec CyFlow Counter and the results were compared with a reference method (FACsCount). A total of 311 blood samples were analysed and the correlation coefficient for the CyFlow Counter was 0.92 (95% CI 0.89-0.95). Mean CD4 counts using the Partec and the reference methods were 308.2 cells/microl and 316.9 cells/microl, respectively. The mean difference in CD4 count values was -8.68 cells/microl (95% CI -18.8 to 1.4). Mean intra-run variation was -6.84 cells/microl (95% CI -12.9 to 0.79). In the district laboratory setting, the instrument could accommodate up to 75 blood samples per technician per day. After being trained, local laboratory staff found the CyFlow Counter procedures simple to run and the instrument easy to manipulate. The Partec CyFlow Counter produces sufficiently reliable results and the instrument appears robust under field conditions. It could provide a new option for introducing routine CD4+ cell monitoring at the district level in the context of scaling-up antiretroviral therapy in Malawi.

Double-structured ultrasonic high frequency reactor using an optimised slant bottom.

The present work has been carried out in order to design a new type of ultrasonic reactor consisting of a double-structured tank. The inner working compartment is built with a slant bottom to allow a better ultrasonic transmission. This paper reports the effect of the inclination angle on acoustic efficiency for several different plates, e.g. two plates made of glass (2 mm and 3 mm thickness) and one made of PVC (3 mm thickness). The acoustic efficiency was determined as the ratio of the signal measured by a hydrophone in the presence of the plate to that signal in the absence of the plate. Having optimised the system, the ultrasonic powers in the inner and the outer compartments of the slant bottom reactor were determined by hydrogen peroxide dosimetry.

Evaluation of methods for the laboratory diagnosis of malaria in Guyana.

When 297 blood samples taken from patients attending a fever clinic in Georgetown Public Hospital were examined microscopically, after thick and thin blood films had been stained with Giemsa, one hundred and forty-two (47.8%) were microscopically positive for malaria. After processing the patient's serum, samples by the Indirect Fluorescent Antibody (IFA) technique, specific IgG and IgM antibodies were detected in 239 (81.3%) and 179 (60.1%), respectively, of the sera. Based on the microscopical findings, the IFAT gave positive predictive and negative values of 54.4% and 81.8% (IgG), and 57.5% and 67.8% (IgM), suggesting that the IgM would be more useful than the IgG in the diagnosis of current malaria. An odds ratio analysis showed that the presence of symptoms, IgG or IgM antibodies, as well as visits to endemic regions, could be good indicators of current malaria. Age and occupation were not. The microscopical method will continue to be the gold standard-the best available criterion for the validation of our tests-for diagnosis of acute malaria.

Evaluation of methods for the laboratory diagnosis of malaria in Guyana

When 297 blood samples taken from patients attending a fever clinic in Georgetowm Public Hospital were examined microscopically, after thick and thin blood films had been stained with Giemsa, one hundred and forty-two (47.8 per cent ) were microscopically positive for malaria. After processing the patients' serum samples by the Indirect Fluourescent Antibody (IFA) technique, specific IgG and IgM antibodies were detected in 239 (81.3 per cent ) and 179 (60.1 per cent ), respectively, of the sera. Based on the microscopical findings, the IFAT gave positive and negative values of 54.4 per cent and 81.8 per cent (IgG), and 57.5 per cent and 67.8 per cent (IgM), suggesting that the IgM would be more useful than the IgG in the diagnosis of current malaria. An odds ratio analysis showed that the presence of symptoms, IgG or IgM antibodies, as well as visits to endemic regions, could be good indicators of current malaria. Age and occupation are not. The microscopical method will continue to be the gold standard - the best available criterion for the validation of our tests - for our diagnosis of acute malaria.

[Comparative studies of Toxocara infections in Belgium and in other countries]. / Etude comparative des infections à Toxocara en Belgique et dans les autres pays.

An enzyme-linked immunosorbent assay with larval stage antigen was used to measure the concentration of antibodies to Toxocara sp in 2206 human sera. The results led to the determination of the specificity of our method, the experimental cutoff estimation and the Toxocara sp seroprevalence in different human populations. The prevalence of canine toxocariasis in pet dogs and the associated environmental contamination were studied through examination of 107 faecal and 20 sand-pits samples for Toxocara sp eggs. Our immunological studies and environmental research revealed the existence of toxocariasis as a zoonotic disease in Belgium. We compare here our results with those found in the world literature: in spite of some surprising results, the environmental factors are important for toxocaral infection. The urban residence, the restricted number of playgrounds for children and areas for dogs, the socioecological status are preponderant in the spread of infection.

Irreversible photolabeling of active site of neutral endopeptidase-24.11 "enkephalinase" by azidothiorphan and [14C]-azidothiorphan.

Azidothiorphan and its [14C]-labeled analogue have been developed as photoaffinity ligands for the active site of the neutral endopeptidase 24.11. In in vitro assays azidothiorphan inhibits the endopeptidase activity with a Ki of 0.75 nM. After ultraviolet irradiation the inhibitor binds irreversibly to the enzyme, and many factors suggest that the photolabeling occurs at the active site. The binding is accompanied by a loss of enzymatic activity, and the inclusion of the competitive inhibitor thiorphan protects the endopeptidase from this inactivation. In addition the binding of another competitive inhibitor [3H]N-[(R,S)-3-hydroxyaminocarbonyl-2-benzyl-1-oxopropyl]-glycine to the active site of endopeptidase-24.11 is inhibited after irradiation with azidothiorphan. Experiments with [14C]-azidothiorphan have shown that very little nonspecific binding of inhibitor to enzyme occurs and the the labeled probe remains bound under denaturing conditions. Azidothiorphan has also been found to produce a long-lasting naloxone-reversible analgesia after intracerebroventricular administration. The results show that azidothiorphan should prove useful both for structural studies and for investigations on the synthesis and turnover of the neutral endopeptidase-24.11.

Pharmacological properties of acetorphan, a parenterally active "enkephalinase" inhibitor.

Acetorphan, i.e. N-[(R,S)-3-acetylmercapto-2-benzylpropanoyl]-glycine, benzyl ester, is a lipophilic derivative of Thiorphan, a potent inhibitor of "enkephalinase" (EC 3.4.24.11). On purified enkephalinase its inhibitory potency was approximately 1000 fold less than that of Thiorphan but became close to the latter (nanomolar) when it was incubated previously with cerebral membranes. After parenteral administration to mice and rats (1-10 mg/kg) extensive inhibition of cerebral enkephalinase was shown by the depressed enzyme activity in brain membranes from treated animals and the long-lasting potentiation of analgesia elicited by (D-Ala2,Met5)enkephalin (i.c.v.). This suggests that acetorphan easily enters the brain where the active Thiorphan is released. Parenteral acetorphan elicited a series of naloxone-reversible, opioid-like effects, most of which were described previously with intracerebral Thiorphan or other enkephalinase inhibitors. Antinociceptive effects were found in some tests (hot plate jump and phenylbenzoquinone-induced writhing) but not in others (hot plate licking and tail withdrawal). "Antidepressant" effect was found in the "mouse despair" test and antidiarrhoeal effect in the rat castor oil test. Acetorphan also elicited significant increases and decreases in turnover indexes of serotonin and noradrenaline, respectively, in mouse cerebral cortex. In mice chronically treated with acetorphan, the antinociceptive activity of the compound was not modified markedly and no overt withdrawal symptom could be observed after either treatment interruption or administration of naloxone.

Dissociated effects of inhibitors of enkephalin-metabolising peptidases or naloxone on various nociceptive responses.

The antinociceptive effects of Thiorphan, an 'enkephalinase' inhibitor, or bestatin, an aminopeptidase inhibitor, as well as of their association and the pronociceptive effects of naloxone, an opiate receptor antagonist, were evaluated in various analgesic tests in mice. These tests could be classified into two groups: (i) those tests in which the two peptidase inhibitors display naloxone-sensitive antinociceptive activity, particularly when administered together, and in which naloxone displays pronociceptive activity (vocalisation, hot-plate jump, writhing), (ii) those tests in which the two peptidase inhibitors and naloxone are ineffective (tail withdrawal, hot-plate licking, tail-flick). In contrast to the above, either morphine or [Met5]enkephalin in subthreshold dosage administrated together with the peptidase inhibitors displayed antinociceptive activity in the two groups of tests. The threshold dosages of morphine were the lowest in tests of the first group. The dissociated and opposite effects of peptidase inhibitors and naloxone per se might reflect a variable participation of endogenous enkephalins (or other opioid peptides) in the control of various nociceptive responses.

New bidentates as full inhibitors of enkephalin-degrading enzymes: synthesis and analgesic properties.

New compounds were designed to fully inhibit the in vitro metabolism of enkephalins, ensured by three different metallopeptidases. For this purpose, bidentate ligands as hydroxamate and N-hydroxy-N-formylamino groups were selected as highly potent metal coordinating agents and introduced on Phe-Gly and Phe-Ala related structures. Compounds corresponding to the general formula HC(O)N(OH)CH2CH(CH2Ph)CONHCH2COOH (compound 7) and HN(OH)C(O)CH2CH(CH2Ph)CONHCH(R)COOH (compound 11, R = H; compound 13, R = CH3) behave as full inhibitors of the three enzymes, with IC50's in the nanomolar range for enkephalinase, from 0.3 microM to 1 nM for dipeptidylaminopeptidase, and in the micromolar range for a biologically relevant aminopeptidase. Two diastereoisomers of the most active inhibitor 13 were separated by HPLC and their stereochemistry was assigned by 1H NMR spectroscopy. Both isomers were efficient as enkephalinase blockers, but only the RS isomer, designated kelatorphan, was able to strongly inhibit aminopeptidase and dipeptidylaminopeptidase. Intracerebroventricular injection in mice of these mixed inhibitors, especially kelatorphan, led to naloxone reversible analgesic responses (hot-plate test) that were slightly better than those produced by a mixture of thiorphan and bestatin, two potent inhibitors of enkephalinase and aminopeptidase, respectively. Kelatorphan was also more efficient in potentiating the analgesia induced by a subanalgesic dose of Met-enkephalin. All these results support a physiological role in pain transmission for enkephalinase and a probably synaptic aminopeptidase M.

Kelatorphan: a full inhibitor of enkephalin degrading enzymes. Biochemical and pharmacological properties, regional distribution of enkephalinase in rat brain by use of a tritiated derivative.

New potent inhibitors of enkephalin degrading enzymes were obtained by synthesis of compounds bearing a bidentate group. Among these bidentates, Kelatorphan, N-[(R)-3-(N-hydroxy)-carboxamido-2-benzylpropanoyl]-L-alanine, is in vitro a full inhibitor of enkephalinase, dipeptidylaminopeptidase and aminopeptidase. In vivo Kelatorphan (i.c.v. administered in mice) prevents exogenous enkephalin from peptidase degradation. The analgesic effect of Kelatorphan is at least equal to that of the association of bestatin and thiorphan. An analogue of Kelatorphan was tritiated and was used as a specific marker of enkephalinase. Thus the distribution of enkephalinase in rat brain was studied: striatum corpus and substantia nigra were particularly labelled.

Simultaneous assessment of the opiate-induced modifications in the vertical and horizontal components of locomotor activity in mice.

The effects of some opiates on the horizontal and vertical components of locomotor activity in mice were measured in actometers fitted with photoelectric cells counting horizontal displacements and the striding over a low partition wall. Morphine and fentanyl, used at low doses which did not modify the horizontal locomotor activity, decreased the striding over of the partition wall. Such a dissociation was not observed with either kappa or sigma agonists which affected in the same way the two components of the locomotor activity: both components were inhibited by kappa agonists and slightly stimulated by the sigma agonist SKF 10047. Although the opiate-induced inhibition of the striding over was induced by low doses of morphine, relatively high doses of naloxone were necessary for its antagonism. This apparently difficult antagonism could result in fact from the sedation which appeared when morphine was associated with naloxone.

Analgesic effects of kelatorphan, a new highly potent inhibitor of multiple enkephalin degrading enzymes.

Kelatorphan, [(R)-3-(N-hydroxy)-carboxamido-2-benzylpropanoyl]-L-alanine, represents the first virtually complete inhibitor of enkephalins metabolism with KI = 1.4 nM against enkephalinase, KI = 2 nM against the Gly2 -Gly3 cleaving dipeptidylaminopeptidase and KI = 7 microM on aminopeptidase activity. The analgesic effect of [Met5]enkephalin was potentiated 50000 times (ED50 approximately 10 ng) by intracerebroventricular co-administration in mice of kelatorphan (50 micrograms). This effect was significantly higher than that produced by bestatin (50 micrograms) + thiorphan (50 micrograms). Kelatorphan alone was at least two-fold more potent as analgesic than the above mixture of inhibitors.

The mu rather than the delta subtype of opioid receptors appears to be involved in enkephalin-induced analgesia.

The analgesic activity of some opioid peptides which display a relative selectivity for either the mu-receptor subtype, [D-Ala2, MePhe4, Gly-ol5]enkephalin (DAGO) or the delta-receptor subtype. [D-Ala2, D-Leu5]enkephalin (DADLE), [D-Ser2, Leu5]enkephalyl-Thr (DSLET) and [D-Thr2, Leu5]enkephalyl-Thr (DTLET) is highly correlated with their affinity for central or peripheral mu- but not delta-receptors. Moreover their analgesic effects as well as those elicited by degrading enzyme inhibitors (bestatin + thiorphan) of endogenous enkephalins were easily antagonized by naloxone with similar pA2 values but not by the delta-antagonist ICI 154,129. Therefore the analgesia produced by opioid peptides including endogenous enkephalins is likely connected to mu-receptor stimulation. Finally, there was no obvious potentiation by delta-agonists of the analgesia resulting from either administration of the mu-agonist morphine or endogenous enkephalins. This suggested that in the hot plate test, there is no modulation of the effect resulting from mu-receptor stimulation by a delta-receptor interaction. Likewise, enkephalinergic activity such as that due to thiorphan + bestatin does not appear to be regulated through mu- or delta-receptor stimulation.

Involvement of endogenous enkephalins in the mouse 'behavioral despair' test.

In the mouse 'behavioral despair' test the immobility time was shortened by [D-Ala2,Met5]enkephalin at doses which did not modify locomotor activity. Similarly, the inhibitors of the enzymes degrading enkephalins, thiorphan and/or bestatin were effective. Their effect was antagonized by naloxone. We conclude that endogenous enkephalins are implicated in the 'behavioral despair' test.

Inhibition of the spontaneous climbing behavior elicited in mice by opiates.

The spontaneous climbing behavior (SCB) developed by naive mice when they are introduced into small individual cylindrical cages with walls of vertical bars is inhibited in a dose-dependent manner by all the tested opiates. Only nalorphine administered over a large scale of doses never completely inhibits the SCB; its maximal effect is only about 50% of total inhibition. The efficacy of opiates on this special component of the locomotor activity has been compared with that on the horizontal displacements measured with an actometer equipped with photoelectric cells. Whereas the drugs known as specific kappa agonists depress both the horizontal locomotor activity and the SCB, most of the other tested opiates inhibit the SCB at doses which do not modify the horizontal locomotion. The low efficacy of loperamide, an opiate agent crossing the blood-brain barrier with difficulty, as well as the strong efficacy of a morphine low dose injected i.c.v. suggest that the opiate-induced SCB inhibition is centrally mediated. Finally, the involvement of mu and kappa receptors is discussed from the observed interactions with various opiate agonists, partial agonist (nalorphine) and antagonists (naloxone and diprenorphine).

Catatonic or hypotonic immobility induced in mice by intracerebroventricular injection of mu or kappa opioid receptor agonists as well as enkephalins or inhibitors of their degradation.

The intracerebroventricular injections in mice of the mu receptor agonists morphine and fentanyl induced an immobility state (the animals staying motionless with the head down on a 45 degree inclined plane) which was apparently hypertonic (catatonia ?) or at least enabled them to remain hanging on a horizontal wire with their forepaws. In similar conditions, injections of the kappa receptor agonists ketocyclazocine and bremazocine induced an immobility state which was hypotonic, in contrast with the preceding one. In a similar way to the mu agonists, Met-enkephalin or Leu-enkephalin injected i.c.v. in association with the inhibitor of enkephalinase thiorphan induced an apparently hypertonic immobility which was easily antagonized by naloxone. The association of thiorphan with bestatin (an inhibitor of aminopeptidases involved in enkephalins inactivation) produced similar results. In contrast, the hypotonic immobility induced by the kappa receptor agonists required relatively high doses of naloxone to be antagonized. The opiate antagonist MR 2266 antagonized equipotent doses of all the above mentioned agents with a similar efficacy. From these data it is suggested that enkephalins could induce an apparently tonic immobility by stimulating mu receptors and that endogenous enkephalins could be involved in a tonic mediation modulating the locomotor activity or regulating the muscular tone.

Complete differentiation between enkephalinase and angiotensin-converting enzyme inhibition by retro-thiorphan.

Thiorphan, N-[(R,S)-3-mercapto-2-benzylpropanoyl]glycine is a highly potent inhibitor (Ki = 3.5 nM) of "enkephalinase," a metalloendopeptidase cleaving the Gly-Phe bond (positions 3 and 4) of enkephalins in brain tissue. In accordance with this property, thiorphan displays antinociceptive activity after systemic administration. However, thiorphan also inhibits to a lesser extent (Ki = 140 nM) the widely distributed angiotensin-converting enzyme, a carboxydipeptidase implicated in blood pressure regulation. Therefore, in view of an eventual clinical use of enkephalinase inhibitors, it was very important to develop fully specific compounds. Such derivatives were obtained taking into account that N-methylation of the ultimate amide bond of dipeptides strongly decreases enkephalinase affinity without affecting angiotension-converting enzyme recognition, whereas retro-inversion of the amide bond leads to the inverse effect. Thus, the retro-inverso dipeptide (R)-H2N-CH(CH2 phi)-NHCO-CH2-CO2H exhibits an inhibitory potency on enkephalinase (IC50 approximately equal to 12 muM) close to that of the natural dipeptide L-Phe-Gly (IC50 approximately equal to 3 muM). This result shows the topological analogy between the crucial components involved in enkephalinase recognition both in active dipeptides and structurally related retro-inverso isomers. Taking into account these observations, retro-thiorphan, (R,S)-HS-CH2-CH-(CH2 phi)-NHCO-CH2-COOH, was prepared. As compared to thiorphan, the retro isomer is 50% as potent (Ki = 6 nM) on enkephalinase but displays a drastic loss of potency on angiotension-converting enzyme (IC50 greater than 10,000 nM). This specificity was interpreted as a consequence of differences in the stereochemical constraints involving enzyme-inhibitor hydrogen bonding. This hypothesis is supported by reported crystallographic studies on related enzymes such as thermolysin and carboxypeptidase A. As expected, retro-thiorphan exhibits about the same analgesic potency as thiorphan on the hot plate and writhing tests in mice. Therefore, the topological concept of retro-inverso isomers could be extended to other enkephalinase inhibitors, allowing the design of potent and highly selective compounds occurring as new classes of analgesic and psychoactive agents.

Inhibition of enkephalin metabolism by, and antinociceptive activity of, bestatin, an aminopeptidase inhibitor.

In the presence of thiorphan an 'enkephalinase' inhibitor, bestatin an aminopeptidase inhibitor of bacterial origin potently inhibited the hydrolysis of [3H][Leu5]enkephalin by slices from rat striatum with an IC50 value of about 0.2 microM whereas puromycin was approximately 1000 times less potent on this preparation. In vivo bestatin or thiorphan (but not puromycin) significantly protected [3H][Met5]enkephalin administered intracerebroventricularly to mice from hydrolysis and co-administration of these two peptidase inhibitors resulted in a strong reduction in the appearance of hydrolysis products in brain. In a parallel fashion the antinociceptive activity of [Met5]enkephalin in the mouse hot-plate test was additively potentiated by bestatin and thiorphan but not by puromycin. Finally both bestatin and thiorphan themselves displayed antinociceptive properties on either the hot-plate jump test or the phenyl-benzo-quinone writhing test. It is concluded that a bestatin-sensitive aminopeptidase activity together with the 'enkephalinase' activity plays a critical role in the inactivation of both exogenous and endogenous enkephalins.