The threat of antimicrobial resistance in developing countries: causes and control strategies.

The causes of antimicrobial resistance (AMR) in developing countries are complex and may be rooted in practices of health care professionals and patients' behavior towards the use of antimicrobials as well as supply chains of antimicrobials in the population. Some of these factors may include inappropriate prescription practices, inadequate patient education, limited diagnostic facilities, unauthorized sale of antimicrobials, lack of appropriate functioning drug regulatory mechanisms, and non-human use of antimicrobials such as in animal production. Considering that these factors in developing countries may vary from those in developed countries, intervention efforts in developing countries need to address the context and focus on the root causes specific to this part of the world. Here, we describe these health-seeking behaviors that lead to the threat of AMR and healthcare practices that drive the development of AMR in developing countries and we discuss alternatives for disease prevention as well as other treatment options worth exploring.

Point-of-care diagnosis and risk factors of infantile, rotavirus-associated diarrhoea in Calabar, Nigeria.

BACKGROUND: Rotaviruses are the primary cause of acute gastroenteritis in young children worldwide and a significant proportion of these infections occur in Africa. OBJECTIVES: In the present study, we determined the prevalence and risk factors of rotavirus infection among children younger than age 5 years with or without diarrhoea in Calabar, Nigeria, using a rapid point-of-care test. METHODS: Two hundred infants younger than age 5 years presenting with acute gastroenteritis and a control group of 200 infants without diarrhoea were tested for rotavirus. Each stool sample was homogenised in an extraction buffer and the supernatant added into the sample well of the Rida Quick rotavirus test cassette and allowed to run for 5 minutes at room temperature. When both the control band and test band were visible on the test cassette a positive result was recorded, whereas when only the control band was visible a negative results was recorded. RESULTS: Rotavirus was detected in 25 (12.5%) of children with diarrhoea and in no children without diarrhoea. Our results demonstrated that children who were exclusively breast-fed by their mothers were not infected with rotavirus and that 92% of the infants infected with rotavirus experienced vomiting. CONCLUSION: These data demonstrate that asymptomatic rotavirus infection is rare and that rotavirus is commonly detected in stool samples of children suffering from diarrhoea with concomitant vomiting. Use of point-of-care rotavirus tests will enhance early diagnosis of rotavirus-associated diarrhoea and reduce irrational use of antibiotics.

Tissue culture drug resistance analysis of a novel HIV-1 protease inhibitor termed PL-100 in non-B HIV-1 subtypes.

PL-100 is a novel HIV-1 protease inhibitor (PI) that maintains activity against viruses that are resistant to other PIs. To further characterize this compound, we used it to select for drug resistance in tissue culture, using two non-B HIV-1 subtypes, viz. subtype C and a CRF01_AE recombinant virus. PL-100 selected for both minor and major PI resistance mutations along either of two distinct pathways. One of these involved the V82A and L90M resistance mutations while the other involved a mutation at position T80I, with other mutations being observed at positions M46I/L, I54M, K55R, L76F, P81S and I85V. The resistance patterns in both subtype C and CRF01_AE were similar and an accumulation of at least three mutations in the flap and active sites were required in each case for high-level resistance to occur, demonstrating that PL-100 has a high genetic barrier against the development of drug resistance.

Development of an allele-specific PCR for detection of the K65R resistance mutation in patients infected with subtype C human immunodeficiency virus type 1.

The selection of drug-resistant variants of human immunodeficiency virus type 1 (HIV-1) is an impediment to the efficiency of antiretroviral (ARV) therapy. We have developed an allele-specific real-time PCR assay to explore the presence of K65R minority species among treated HIV-1 subtype B and C infections. Thirty HIV-1 subtype C- and 26 subtype B-infected patients lacking K65R as determined by conventional sequencing methods were studied, and viral minority species were found in four HIV-1 subtype C samples.

Effects of the K65R and K65R/M184V reverse transcriptase mutations in subtype C HIV on enzyme function and drug resistance.

BACKGROUND: We investigated the effects of mutations K65R and K65R plus M184V on enzymatic function and mechanisms of drug resistance in subtype C reverse transcriptase (RT). METHODS: Recombinant subtype C HIV-1 RTs containing K65R or K65R+M184V were purified from Escherichia coli. Enzyme activities and tenofovir (TFV) incorporation efficiency by wild-type (WT) and mutant RTs of both subtypes were determined in cell-free assays. Efficiency of (-) ssDNA synthesis and initiation by subtype C RTs was measured using gel-based assays with HIV-1 PBS RNA template and tRNA3Lys as primer. Single-cycle processivity was assayed under variable dNTP concentrations. Steady-state analysis was performed to measure the relative inhibitory capacity (ki/km) of TFV-disphosphate (TFV-DP). ATP-dependent excision and rescue of TFV-or ZDV-terminated DNA synthesis was monitored in time-course experiments. RESULTS: The efficiency of tRNA-primed (-)ssDNA synthesis by subtype C RTs was: WT > K65R > K65R+M184V RT. At low dNTP concentration, K65R RT exhibited lower activity in single-cycle processivity assays while the K65R+M184V mutant showed diminished processivity independent of dNTP concentration. ATP-mediated excision of TFV-or ZDV-terminated primer was decreased for K65R and for K65R+M184V RT compared to WT RT. K65R and K65R+M184V displayed 9.8-and 5-fold increases in IC50 for TFV-DP compared to WT RT. The Ki/Km of TFV was increased by 4.1-and 7.2-fold, respectively, for K65R and K65R+M184V compared to WT RT. CONCLUSION: The diminished initiation efficiency of K65R-containing RTs at low dNTP concentrations have been confirmed for subtype C as well as subtype B. Despite decreased excision, this decreased binding/incorporation results in diminished susceptibility of K65R and K65R+M184 RT to TFV-DP.

Detection of human immunodeficiency virus (HIV) type 1 M184V and K103N minority variants in patients with primary HIV infection.

We used an allele-specific real-time PCR assay to explore the presence of K103N and M184V minority species among primary human immunodeficiency virus (HIV) infections and their potential influence in HIV transmission. Thirty randomly chosen antiretroviral drug-naive patients lacking both the K103N and the M184V mutations as determined by conventional sequencing methods were studied, and K103N and M184V viral minority species were found in three (10%) and four (11%) patients, respectively.

Nucleoside and nucleotide analogs select in culture for different patterns of drug resistance in human immunodeficiency virus types 1 and 2.

Recent findings suggest bidirectional antagonisms between the K65R mutation and thymidine analogue mutations in human immunodeficiency virus type 1 (HIV-1)-infected, treatment-experienced patients, yet little is known about HIV-2 in this regard. This study addressed the effects of innate polymorphisms in HIV-2 on emergent resistance to nucleoside/nucleotide analogues. Emergent drug resistance profiles in HIV-2 subtypes A (n = 3) and B (n = 1) were compared to those of HIV-1 subtypes B and C. Drug resistance was evaluated with cord blood mononuclear cells (CBMCs) and MT2 cells, using selective pressure with tenofovir (TFV), zidovudine (ZDV), stavudine (d4T), didanosine (ddI), abacavir (ABC), lamivudine (3TC), emtricitabine (FTC), or various dual-drug combinations. Resistance was evaluated using conventional and ultrasensitive sequencing approaches. In agreement with our previous findings, dual-drug combinations of TFV, ddI, ABC, d4T, ZDV, and 3TC preferentially selected for K65R in HIV-1 subtype C isolates. In HIV-1 subtype B, TFV-3TC and ZDV-3TC selected for M184I and D67N, respectively. In contrast, selections with all four HIV-2 cultures favored the development of M184I in dual-drug combinations that included either 3TC or FTC. Since HIV-2 cultures did not develop K65R, an ultrasensitive allele-specific real-time PCR assay was developed to distinguish the presence of 65R from wild-type K65 after 16 cycles with a discriminatory ability of 0.1% against a population of wild-type virus. These results underscore potential differences in emergent drug resistance pathways in HIV-1 and HIV-2 and show that polymorphisms may influence the development of the resistance pathways that are likely to emerge.

Benefits of an educational program for journalists on media coverage of HIV/AIDS in developing countries.

OBJECTIVE: a) To assess the suitability of the curriculum content and didactical quality of information delivered to educate journalists in the J2J program in HIV/AIDS (process evaluation) and b) to explore the effects of such programs on journalists' reporting of HIV/AIDS related information (outcome evaluation). DESIGN: Descriptive study. METHODS: For the process evaluation, each J2J program curriculum was evaluated for accuracy and pertinence by individuals with high familiarity with HIV/AIDS research. For the outcome evaluation, a survey of J2J attendees and evaluations of the program lectures by attendees were performed in chronological order to determine their perception on usefulness of the program. RESULTS: Overall, the J2J curriculum is successful in providing journalists with a clear understanding of the current HIV/AIDS medical research objectives and issues with most journalists reporting an increased ability to better investigate and disseminate accurate information on this subject. Furthermore, the journalists surveyed reported positive community responses directly as a result of the J2J training. CONCLUSION: The J2J program helps to increase global awareness of pertinent HIV/AIDS concepts. Through this professional development strategy, journalists from around the world may help to amplify efforts to prevent new HIV infections and quench the dissemination of inaccurate information and folklore.

Discrepancies in assignment of subtype/recombinant forms by genotyping programs for HIV type 1 drug resistance testing may falsely predict superinfection.

With the growing diversity of the HIV pandemic, routine genotyping is an important tool for monitoring viral subtype as well as drug resistance. In this regard, numerous subtyping tools and drug resistance algorithms are available online. However, there are discrepancies in the use of these online tools in the designation of HIV-1 subtypes or recombinant forms that may have an impact on drug susceptibility profiles. Indeed, inconsistencies in some of these tools may lead to a false designation of dual infection and/or superinfection. In this case study, we evaluated the sequence diversity of an infection that was referred to us as a potential case of superinfection as a result of variations in designation of subtype. We evaluated sequences using five different online tools and finally determined by phylogenetic analysis that the sequence was a unique A1/C intersubtype recombinant at baseline and not a case of superinfection.

Near full-length genomic analysis of a novel subtype A1/C recombinant HIV type 1 isolate from Canada.

The rapid introduction of non-B HIV-1 subtypes into Quebec, mostly from persons from regions where HIV prevalence is high and where different variants circulate, means that recombination must now be an important consideration in the epidemiologic surveillance of HIV infection. No circulating recombinant form (CRF), currently involving exclusively subtypes A1 and C, exists in the Los Alamos HIV database. This study presents a near full-length genomic analysis of a novel HIV-1 recombinant involving subtypes A1 and C. Bootscanning revealed that the recombinant structure involves three breakpoints that separate the genome into four regions, alternating between subtypes A1 and C. The intersubtype recombinant breakpoint in the pol gene was at midpoint between the protease and reverse transcriptase open reading frames. This is the first report of a recombinant involving subtypes A1 and C in Canada, the epidemiologic significance of which is not yet known.

Variations in reverse transcriptase and RNase H domain mutations in human immunodeficiency virus type 1 clinical isolates are associated with divergent phenotypic resistance to zidovudine.

Mutations in the RNase H domain of human immunodeficiency virus type 1 RT have been reported to cause resistance to zidovudine (ZDV) in vitro. However, very limited data on the in vivo relevance of these mutations in patients exist to date. This study was designed to determine the relationship between mutations in the RNase H domain and viral susceptibility to nucleoside analogues. Viruses harboring complex thymidine analogue mutation (TAM) and nucleoside analogue mutation (NAM) profiles were evaluated for their phenotypic susceptibilities to ZDV, tenofovir (TNF), and the nonapproved nucleoside reverse transcriptase inhibitors (NRTIs) beta-2',3'-didehydro-2',3'-dideoxy-5-fluorocytidine (Reverset), beta-D-5-fluorodioxolane-cytosine, and apricitabine. As controls, viruses from NRTI-naïve patients were also studied. The pol RT region (codons 21 to 250) of the viruses were sequenced and evaluated for mutations in the RNase H domain (codons 441 to 560) and the connection domain (codons 289 to 400). The results showed that viruses from patients failing multiple NRTI-containing regimens had distinct TAM and NAM profiles that conferred various degrees of resistance to ZDV (0.9- to >300-fold). Sequencing of the RNase H domain identified five positions (positions 460,468, 483, 512, and 519) at which extensive amino acid polymorphisms common in both wild-type viruses and viruses from treated patients were identified. No mutations were observed at positions 539 and 549, which have previously been associated with ZDV resistance. Mutations in the RNase H domain did not appear to correlate with the levels of phenotypic resistance to ZDV. Although some mutations were also observed in the connection domain, the simultaneous presence of the L74V and M184V mutations was the most significant determinant of phenotypic resistance to ZDV in patients infected with viruses with TAMs.

High rates of forward transmission events after acute/early HIV-1 infection.

BACKGROUND: A population-based phylogenetic approach was used to characterize human immunodeficiency virus (HIV)-transmission dynamics in Quebec. METHODS: HIV-1 pol sequences included primary HIV infections (PHIs; <6 months after seroconversion) from the Quebec PHI cohort (1998-2005; n=215) and the provincial genotyping program (2001-2005; n=481). Phylogenetic analysis determined sequence interrelationships among unique PHIs (n=593) and infections from untreated (n=135) and treated (n=660) chronically infected (CI) potential transmitter populations (2001-2005). Clinical features, risk factors, and drug resistance for clustered and nonclustered transmission events were ascertained. RESULTS: Viruses from 49.4% (293/593) of PHIs cosegregated into 75 transmission chains with 2-17 transmissions/cluster. Half of the clusters included 2.7+/-0.8 (mean+/-SD) transmissions, whereas the remainder had 8.8+/-3.5 transmissions. Maximum periods for onward transmission in clusters were 15.2+/-9.5 months. Coclustering of untreated and treated CIs with PHIs were infrequent (6.2% and 4.8%, respectively). The ages, viremia, and risk factors were similar for clustered and nonclustered transmission events. Low prevalence of drug resistance in PHI supported amplified transmissions at early stages. CONCLUSIONS: Early infection accounts for approximately half of onward transmissions in this urban North American study. Therapy at early stages of disease may prevent onward HIV transmission.

Natural polymorphisms in the human immunodeficiency virus type 2 protease can accelerate time to development of resistance to protease inhibitors.

Human immunodeficiency virus type 2 (HIV-2) contains numerous natural polymorphisms in its protease (PR) gene that are implicated in drug resistance in the case of HIV-1. This study evaluated emergent PR resistance in HIV-2. Three HIV-2 isolates were selected for resistance to amprenavir (APV), nelfinavir (NFV), indinavir (IDV), and tipranavir (TPV) in cell culture. Genotypic analysis determined the time to the appearance of protease inhibitor (PI)-associated mutations compared to HIV-1. Phenotypic drug susceptibility assays were used to determine the levels of drug resistance. Within 10 to 15 weeks of serial passage, three major mutations--I54M, I82F, and L90M--arose in HIV-2 viral cultures exposed to APV, NFV, and IDV, whereas I82L was selected with TPV. After 25 weeks, other cultures had developed I50V and I84V mutations. In contrast, no major PI mutations were selected in HIV-1 over this period except for D30N in the context of NFV selective pressure. The baseline phenotypes of wild-type HIV-2 isolates were in the range observed for HIV-1, except for APV and NFV for which a lower degree of sensitivity was seen. The acquisition of the I54M, I84V, L90M, and L99F mutations resulted in multi-PI-resistant viruses, conferring 10-fold to more than 100-fold resistance. Of note, we observed a 62A/99F mutational motif that conferred high-level resistance to PIs, as well as novel secondary mutations, including 6F, 12A, and 21K. Thus, natural polymorphisms in HIV-2 may facilitate the selection of PI resistance. The increasing incidence of such polymorphisms in drug-naive HIV-1- and HIV-2-infected persons is of concern.

HIV-1 subtype C viruses rapidly develop K65R resistance to tenofovir in cell culture.

BACKGROUND: Genotypic diversity among HIV-1 subtypes and circulating recombinant forms (CRF) may lead to distinct pathways to drug resistance. This study evaluated subtype-related differences in the development of resistance in culture to tenofovir. METHODS: Genotyping determined nucleotide diversity among subtypes. Representative subtype B, C, CRF1_AE, CRF2_AG, G, and HIV-2 isolates were selected for resistance to tenofovir, lamivudine and didanosine in cell culture. Phenotypic assays determined the effects of the K65R substitution in reverse transcriptase (RT) on drug susceptibility. RESULTS: Subtype C isolates show unique polymorphisms in RT codons 64 (AAG-->AAA), 65 (AAA-->AAG), and 66 (AAA-->AAG), absent in other subtypes. The K65R mutation (AAG-->AGG) arose with tenofovir by week 12 in four subtype C selections. In contrast, no tenofovir resistance arose in four subtype B (> 34-74 weeks), one each of CRF2_AG and G (> 30-33 weeks), and three HIV-2 (> 27-28 weeks) selections. K65R appeared after 55 and 73 weeks in two CRF1_AE selections with tenofovir. In contrast, times to the appearance of M184V with lamivudine pressure (weeks 8-14) did not vary among subtypes. Selective didanosine pressure resulted in the appearance of M184V and L74V after 38 weeks in two of four subtype C selections. The K65R transitions in subtype C and other subtypes (AGG and AGA) conferred similar 6.5-10-fold resistance to tenofovir and five to 25-fold cross-resistance to each of abacavir, lamivudine, and didanosine, while not affecting zidovudine susceptibility. CONCLUSION: Tenofovir -based regimens will need to be carefully monitored in subtype C infections for the possible selection of K65R.

Neutralization kinetics of sensitive and resistant subtype B primary human immunodeficiency virus type 1 isolates.

The aim of the study was to determine if sensitive and resistant human immunodeficiency virus type 1 (HIV-1) subtype B primary isolates have different neutralization kinetics. Neutralization assays were undertaken where either the time allowed for virus to react with antibodies or the subsequent period of this mixture's exposure to target cells were varied. The relative neutralization sensitivity/resistance is a reproducible property of the isolates. In a minority of combinations, the titre falls exponentially for as long as the free virions are exposed to antibody. In the remainder, neutralization kinetics shows deviations which may be attributed to events occurring after the virus-antibody mixture is added to the target cells: significant neutralization with minimal exposure of the free virions to antibody; a plot where reduction in virus titre is parallel to the duration of the incubation phase of the assay. Neutralization rate constants are similar for primary HIV-1 SF33, HIV-1 SF162, and HIV-1 89.6, reaching 5 x 10(5)-1 x 10(6)/M sec for the monoclonal antibody IgG1 b12. However, although increased antibody levels produced greater reductions in virus titre the rate of neutralization was not proportional to the antibody concentration. Neutralization of either the free virion or cell-associated virus does not correlate with the resistance/sensitivity of primary subtype B isolates. The target cells play an active role, so that in designing neutralization assays with primary isolates of HIV-1, events following the virus-antibody complex binding to the cell surface have to be taken into consideration.