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.

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.

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.

New carboxyalkyl inhibitors of brain enkephalinase: synthesis, biological activity, and analgesic properties.

New carboxyalkyl compounds derived from Phe-Leu and Phe-Ala were synthesized and checked as inhibitors of "enkephalinase", a metalloendopeptidase cleaving the Gly3-Phe4 bond of enkephalins from mouse striatal membranes. Differential recognition of both brain enkephalinase and angiotensin-converting enzyme (ACE) catalytic sites by these carboxylalkyl compounds lead to potent (KI approximately 0.5 microM), competitive and selective inhibitors of the enkephalin-degrading enzyme. The most interesting compound, N-[(RS)-2-carboxy-3-phenylpropanoyl]-L-leucine (3, KI = 0.34 microM), is 10000 times more potent on enkephalinase than on ACE activities. Intracerebroventricular (icv) injection of 3 in mice leads to a high potentiation of the analgesic effect of the exogenously administered D-Ala2-Met-enkephalin, evidencing the in vivo inhibition of enkephalinase. Moreover, icv administration of 3 alone induces a dose-dependent analgesia in mice measured on both hot-plate and writhing tests. In the former assay, the ED50 was approximately 10 micrograms per animal, slightly higher than that of thiorphan. All the antinociceptive effects were antagonized by naloxone, demonstrating the involvement of enkephalins in analgesia and their in vivo protection from enkephalinase by 3. The described compounds can be considered as first examples of a new series of analgesics and potentially psychoactive agents.

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.

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.

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 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.

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.

Enkephalin metabolism in brain and its inhibition.

Various cerebral peptidases are able to hydrolyse the enkephalins into inactive fragments. Among these enzymes enkephalin-dipeptidylcarboxypeptidase ("enkephalinase") inhibited by thiorphan and a bestatin-sensitive aminopeptidase activity seem to play a key-role as "inactivating neuropeptidases". Their inhibition, in vitro as well as in vivo, leads to a protection of endogenous enkephalins and to antinociceptive effects.

Peptidases involved in the inactivation of exogenous and endogenous enkephalins.

Among the various cerebral enzyme activities able to hydrolyse the enkephalins into inactive fragments only two seem responsible for the metabolism of the endogenous opioid peptides: a dipeptidylcarboxypeptidase ("enkephalinase"), and a bestatin-sensitive aminopeptidase. Their inhibition by thiorphan and bestatin results in an antinociceptive effect observed in tests in which the nociceptive stimulation is probably accompanied by a concomittent release of enkephalins.

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.

Pain control by endogenous enkephalins is mediated by mu opioid receptors.

The analgesic effects of bestatin and thiorphan, two enzymatic inhibitors protecting endogenous enkephalins from their degradation, and those of DAGO and deltakephalin, respectively mu and delta opioid peptides, are assessed on the electrical stimulation test of the mouse tail. The relative analgesic potency of DAGO and deltakephalin is in good agreement with their relative potency on mu pharmacological assays: inhibition of electrically-induced contractions of guinea-pig ileum, displacement of 3H DAGO on rat brain. Finally, the analgesic effects of DAGO, deltakephalin and bestatin + thiorphan, are antagonized by the mu antagonist naloxone with similar pA2, and they are not modified by the delta antagonist ICI 154, 129. We conclude that only mu and not delta receptors are involved in the analgesic effects of enkephalins.

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.

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.