Crystalloid resuscitation is associated with decreased treatment delays and improved systolic blood pressures in a blood-constrained setting.

Objectives: We analyzed resuscitation practices in Cameroonian patients with trauma as a first step toward developing a context-appropriate resuscitation protocol. We hypothesized that more patients would receive crystalloid-based (CB) resuscitation with a faster time to administration than blood product (BL) resuscitation. Methods: We included patients enrolled between 2017 and 2019 in the Cameroon Trauma Registry (CTR). Patients presenting with hemorrhagic shock (systolic blood pressure (SBP) <100 mm Hg and active bleeding) were categorized as receiving CB, BL, or no resuscitation (NR). We evaluated differences between cohorts with the Kruskal-Wallis test for continuous variables and Fisher's exact test for categorical variables. We compared time to treatment with the Wilcoxon rank sum test. Results: Of 9635 patients, 403 (4%) presented with hemorrhagic shock. Of these, 278 (69%) patients received CB, 39 (10%) received BL, and 86 (21%) received NR. BL patients presented with greater injury severity (Highest Estimated Abbreviated Injury Scale (HEAIS) 4 BL vs 3 CB vs 1 NR, p<0.001), and lower median hemoglobin (8.0 g/dL BL, 11.4 g/dL CB, 10.6 g/dL NR, p<0.001). CB showed greater initial improvement in SBP (12 mm Hg CB vs 9 mm Hg BL vs 0 NR mm Hg, p=0.04) compared with BL or no resuscitation, respectively. Median time to treatment was lower for CB than BL (12 vs 131 min, p<0.01). Multivariate logistic regression adjusted for injury severity found no association between resuscitation type and mortality (CB adjusted OR (aOR) 1.28, p=0.82; BL aOR 1.05, p=0.97). Conclusions: CB was associated with faster treatment, greater SBP elevation, and similar survival compared with BL in Cameroonian patients with trauma with hemorrhagic shock. In blood-constrained settings, treatment delays associated with blood product transfusion may offset the physiologic benefits of an early BL strategy. CB prior to definitive hemorrhage control in this resource-limited setting may be a necessary strategy to optimize perfusion pressure. Level of evidence and study type: III, retrospective study.

The CBS test: Development, evaluation & cross-validation of a community-based injury severity scoring system in Cameroon.

Injury-related deaths overwhelmingly occur in low and middle-income countries (LMICs). Community-based injury surveillance is essential to accurately capture trauma epidemiology in LMICs, where one-third of injured individuals never present to formal care. However, community-based studies are constrained by the lack of a validated surrogate injury severity metric. The primary objective of this bipartite study was to cross-validate a novel community-based injury severity (CBS) scoring system with previously-validated injury severity metrics using multi-center trauma registry data. A set of targeted questions to ascertain injury severity in non-medical settings-the CBS test-was iteratively developed with Cameroonian physicians and laypeople. The CBS test was first evaluated in the community-setting in a large household-based injury surveillance survey in southwest Cameroon. The CBS test was subsequently incorporated into the Cameroon Trauma Registry, a prospective multi-site national hospital-based trauma registry, and cross-validated in the hospital setting using objective injury metrics in patients presenting to four trauma hospitals. Among 8065 surveyed household members with 503 injury events, individuals with CBS indicators (CBS+) were more likely to report ongoing disability after injury compared to CBS- individuals (OR 1.9, p = 0.004), suggesting the CBS test is a promising injury severity proxy. In 9575 injured patients presenting for formal evaluation, the CBS test strongly predicted death in patients after controlling for age, sex, socioeconomic status, and injury type (OR 30.26, p<0.0001). Compared to established injury severity scoring systems, the CBS test comparably predicts mortality (AUC: 0.8029), but is more feasible to calculate in both the community and clinical contexts. The CBS test is a simple, valid surrogate metric of injury severity that can be deployed widely in community-based surveys to improve estimates of injury severity in under-resourced settings.

How trauma patients die in low resource settings: Identifying early targets for trauma quality improvement.

BACKGROUND: Injury deaths in sub-Saharan Africa are among the world's highest, but hospital data rarely have sufficient granularity to direct quality improvement. We analyzed clinical care patterns among trauma patients who died in a prospective, multicenter sub-Saharan cohort to pinpoint trauma quality improvement intervention targets. METHODS: In-hospital trauma deaths in four Cameroonian hospitals between 2017 and 2019 were included. Trauma registry data on patient demographics, injury characteristics, and clinical care were analyzed to identify opportunities for systems improvements. RESULTS: Among 9,423 trauma patients, there were 236 deaths. Overall, 83% of patients who died in the emergency department were living on arrival (LOA). Among 183 LOA patients, 30% presented with normal vital signs, but 11% had no vital signs taken, often because of lack of equipment (43%). Of LOA patients presenting with a Glasgow Coma Scale score of <9 (56%), few received neurosurgery consults (15%), C-collar placement (9%), or intubation (1%). The most common reason for lack of c-collar placement was failure to recognize that it was indicated (66%). Tracheal deviation, unequal breath sounds, or paradoxical chest movement were present in 63% of LOA patients, but only two patients had chest tubes placed. Hypotension or active bleeding was present in 80% of LOA patients; while crystalloid bolus was given to 96% of these patients, few received transfusion (8%), tourniquet placement for extremity injury (6%), or an operation (4%). CONCLUSION: Primary survey interventions are underperformed in trauma nonsurvivors in Cameroon. Protocolizing early treatment for head injury, hemorrhagic shock, and chest wall trauma could reduce trauma mortality. LEVEL OF EVIDENCE: Prognostic and Epidemiologic; Level III.

Chronic Disease Comorbidities Among Injured Patients in Cameroon: A Retrospective Cohort Study.

INTRODUCTION: Chronic diseases are increasing but underdiagnosed in low-income and middle-income countries (LMICs), where injury mortality is already disproportionately high. We estimated prevalence of known chronic disease comorbidities and their association with outcomes among injured patients in Cameroon. MATERIALS AND METHODS: Injured patients aged ≥15 y presenting to four Cameroonian hospitals between October 2017 and January 2020 were included. Our explanatory variable was known chronic disease; prevalence was age-standardized. Outcomes were overall in-hospital mortality and admission or transfer from the emergency department (ED). Associations between known chronic disease and outcomes were evaluated using logistic regression adjusted for age, gender, estimated injury severity score (eISS), hospital, and household socioeconomic status. Unadjusted eISS-stratified and age-stratified outcomes were also compared via chi-squared tests. RESULTS: Of 7509 injured patients, 370 (4.9%) reported at least one known chronic disease; age-standardized prevalence was 8.4% (95% confidence interval [CI] 7.5%-9.2%). Patients with known chronic disease had higher mortality (4.6% versus 1.5%, adjusted odds ratio [aOR]: 2.61 [95% CI: 1.25-5.47], P = 0.011) and were more likely to be admitted or transferred from the ED (38.7% versus 19.8%, aOR: 1.40 [95% CI: 1.02-1.92], P = 0.038) compared to those without known comorbidities. Crude differences in mortality (11.3% versus 3.3%, P = 0.002) and hospital admission or transfer (63.8% versus 46.6%, P = 0.011) were most notable for patients with eISS 16-24. CONCLUSIONS: Despite underdiagnosis among Cameroonians, we demonstrated worse injury outcomes among those with known chronic diseases. Integrating chronic disease screening with injury care may help address underdiagnosis in Cameroon. Future work should assess whether chronic disease prevention in LMICs could improve injury outcomes.

Associations between social determinants of health and interpersonal violence-related injury in Cameroon: a cross-sectional study.

INTRODUCTION: Risk factors for interpersonal violence-related injury (IPVRI) in low-income and middle-income countries (LMICs) remain poorly defined. We describe associations between IPVRI and select social determinants of health (SDH) in Cameroon. METHODS: We conducted a cross-sectional analysis of prospective trauma registry data collected from injured patients >15 years old between October 2017 and January 2020 at four Cameroonian hospitals. Our primary outcome was IPVRI, compared with unintentional injury. Explanatory SDH variables included education level, employment status, household socioeconomic status (SES) and alcohol use. The EconomicClusters model grouped patients into household SES clusters: rural, urban poor, urban middle-class (MC) homeowners, urban MC tenants and urban wealthy. Results were stratified by sex. Categorical variables were compared via Pearson's χ2 statistic. Associations with IPVRI were estimated using adjusted odds ratios (aOR) with 95% confidence intervals (95%CI). RESULTS: Among 7605 patients, 5488 (72.2%) were men. Unemployment was associated with increased odds of IPVRI for men (aOR 2.44 (95% CI 1.95 to 3.06), p<0.001) and women (aOR 2.53 (95% CI 1.35 to 4.72), p=0.004), as was alcohol use (men: aOR 2.33 (95% CI 1.91 to 2.83), p<0.001; women: aOR 3.71 (95% CI 2.41 to 5.72), p<0.001). Male patients from rural (aOR 1.45 (95% CI 1.04 to 2.03), p=0.028) or urban poor (aOR 2.08 (95% CI 1.27 to 3.41), p=0.004) compared with urban wealthy households had increased odds of IPVRI, as did female patients with primary-level/no formal (aOR 1.78 (95% CI 1.10 to 2.87), p=0.019) or secondary-level (aOR 1.54 (95% CI 1.03 to 2.32), p=0.037) compared with tertiary-level education. CONCLUSION: Lower educational attainment, unemployment, lower household SES and alcohol use are risk factors for IPVRI in Cameroon. Future research should explore LMIC-appropriate interventions to address SDH risk factors for IPVRI.

Evaluation of corrective actions of feedback from clinicians on Clinical Laboratory Services at Bamenda Regional Hospital Laboratory, Cameroon.

BACKGROUND: Customers' satisfaction is imperative for success. Clinical laboratories continuously strive to attain very high levels of customer satisfaction to serve their clients and maintain accreditation. The concept of customer satisfaction has not yet been asserted in most clinical laboratories in Cameroon. OBJECTIVES: Our objectives were to assess the satisfaction of clinicians with the laboratory services at the Bamenda Regional Hospital Laboratory, identify important challenges, corrective actions implemented and changes in satisfaction. METHODS: This retrospective study reviewed secondary data from clinician satisfaction survey records from March 2017 and November 2017. Challenges and implemented corrective actions were identified for assessed statements of dissatisfaction (dissatisfaction rates ≥ 20%) on the March 2017 survey. Satisfaction rates in March 2017 and November 2017 were compared. RESULTS: High levels of dissatisfaction were observed for general satisfaction, waiting time, communication, duty consciousness, specimen collection and approach on the March 2017 survey. The main challenges identified were: lack of respect for the expected length of the waiting time, poor attitude, inadequate information, staff shortage and inadequate supervision. Statistically significant reductions in rates of dissatisfaction were observed for general satisfaction, waiting time, communication, response to emergencies, issuing of results, specimen collection, approach and duty consciousness. CONCLUSION: Waiting time is a major cause of clinician dissatisfaction with laboratory services. The identification of clinicians' challenges and the effective implementation of corrective actions contribute to improvements in clinician satisfaction.

Safety, reactogenicity, and immunogenicity of a chimpanzee adenovirus vectored Ebola vaccine in adults in Africa: a randomised, observer-blind, placebo-controlled, phase 2 trial.

BACKGROUND: The 2014 Zaire Ebola virus disease epidemic accelerated vaccine development for the virus. We aimed to assess the safety, reactogenicity, and immunogenicity of one dose of monovalent, recombinant, chimpanzee adenovirus type-3 vectored Zaire Ebola glycoprotein vaccine (ChAd3-EBO-Z) in adults. METHODS: This phase 2, randomised, observer-blind, controlled trial was done in study centres in Cameroon, Mali, Nigeria, and Senegal. Healthy adults (≥18 years) were randomly assigned with a web-based system (1:1; minimisation procedure accounting for age, gender, centre) to receive ChAd3-EBO-Z (day 0), or saline placebo (day 0) and ChAd3-EBO-Z (month 6). The study was observer-blind until planned interim day 30 analysis, single-blind until month 6, and open-label after month 6 vaccination. Primary outcomes assessed in the total vaccinated cohort, which comprised all participants with at least one study dose administration documented, were serious adverse events (up to study end, month 12); and for a subcohort were solicited local or general adverse events (7 days post-vaccination), unsolicited adverse events (30 days post-vaccination), haematological or biochemical abnormalities, and clinical symptoms of thrombocytopenia (day 0-6). Secondary endpoints (subcohort; per-protocol cohort) evaluated anti-glycoprotein Ebola virus antibody titres (ELISA) pre-vaccination and 30 days post-vaccination. This study is registered with ClinicalTrials.gov, NCT02485301. FINDINGS: Between July 22, 2015, and Dec 10, 2015, 3030 adults were randomly assigned; 3013 were included in the total vaccinated cohort (1509 [50·1%] in the ChAd3-EBO-Z group and 1504 [49·9%] in the placebo/ChAd3-EBO-Z group), 17 were excluded because no vaccine was administered. The most common solicited injection site symptom was pain (356 [48%] of 748 in the ChAd3-EBO-Z group vs 57 [8%] of 751 in the placebo/ChAd3-EBO-Z group); the most common solicited general adverse event was headache (345 [46%] in the ChAd3-EBO-Z group vs 136 [18%] in the placebo/ChAd3-EBO-Z group). Unsolicited adverse events were reported by 123 (16%) of 749 in the ChAd3-EBO-Z group and 119 (16%) of 751 in the placebo/ChAd3-EBO-Z group. Serious adverse events were reported for 11 (1%) of 1509 adults in the ChAd3-EBO-Z group, and 18 (1%) of 1504 in the placebo/ChAd3-EBO-Z group; none were considered vaccination-related. No clinically meaningful thrombocytopenia was reported. At day 30, anti-glycoprotein Ebola virus antibody geometric mean concentration was 900 (95% CI 824-983) in the ChAd3-EBO-Z group. There were no treatment-related deaths. INTERPRETATION: ChAd3-EBO-Z was immunogenic and well tolerated in adults. Our findings provide a strong basis for future development steps, which should concentrate on multivalent approaches (including Sudan and Marburg strains). Additionally, prime-boost approaches should be a focus with a ChAd3-based vaccine for priming and boosted by a modified vaccinia Ankara-based vaccine. FUNDING: EU's Horizon 2020 research and innovation programme and GlaxoSmithKline Biologicals SA.

Safety, reactogenicity, and immunogenicity of a chimpanzee adenovirus vectored Ebola vaccine in children in Africa: a randomised, observer-blind, placebo-controlled, phase 2 trial.

BACKGROUND: During the large 2013-16 Ebola virus outbreak caused by the Zaire Ebola virus, about 20% of cases were reported in children. This study is the first, to our knowledge, to evaluate an Ebola vaccine in children younger than 6 years. We aimed to evaluate the safety, reactogenicity, and immunogenicity of a monovalent, recombinant, chimpanzee adenovirus type-3 vectored Zaire Ebola glycoprotein vaccine (ChAd3-EBO-Z) in a paediatric population. METHODS: This phase 2, randomised, observer-blind, controlled trial was done in a vaccine centre in Mali and a university hospital centre in Senegal. Healthy children were randomly assigned through a web-based system (1:1; stratified by age group, gender, and centre) to receive ChAd3-EBO-Z (day 0) and meningococcal serogroups A,C,W-135,Y tetanus toxoid conjugate vaccine (MenACWY-TT; month 6), or MenACWY-TT (day 0) and ChAd3-EBO-Z (month 6). The study was observer-blind from study start until interim day 30 analysis and became single-blind as of interim analysis. Primary outcomes assessed were serious adverse events (up to study end, month 12), solicited local or general adverse events (7 days post-vaccination), unsolicited adverse events (30 days post-vaccination), haematological or biochemical abnormalities, and clinical symptoms of thrombocytopenia (day 0-6). As secondary endpoints, we evaluated anti-glycoprotein Zaire Ebola virus antibody titres (ELISA) pre-vaccination and 30 days post-vaccination. This study is registered with ClinicalTrials.gov, NCT02548078. FINDINGS: From Nov 11, 2015, to May 9, 2016, of 776 children screened for eligibility, 600 were randomly assigned (200 [33%] in each age strata: 1-5, 6-12, 13-17 years), 300 (50%) to the ChAd3-EBO-Z/MenACWY-TT group and 300 (50%) to the MenACWY-TT/ChAd3-EBO-Z group; all were included in the total vaccinated cohort. Post-day 0 vaccination, the most common solicited injection site symptom was pain (127 [42%] of 300 in the ChAd3-EBO-Z/MenACWY-TT group vs 60 [20%] of 300 in the MenACWY-TT/ChAd3-EBO-Z group); the most common solicited general adverse event was fever (95 [32%] of 300 in the ChAd3-EBO-Z/MenACWY-TT group vs 28 [9%] of 300 in the MenACWY-TT/ChAd3-EBO-Z group). Unsolicited adverse events post-day 0 vaccination were reported by 41 (14%) of 300 participants in the ChAd3-EBO-Z/MenACWY-TT group and 24 (8%) of 300 MenACWY-TT/ChAd3-EBO-Z recipients. Serious adverse events were reported for two (1%) of 300 children in each group; none were considered vaccination related. No clinical symptoms of thrombocytopenia were reported. At day 30, anti-glycoprotein Ebola virus antibody geometric mean concentrations (GMC) in the ChAd3-EBO-Z/MenACWY-TT group were 1564 (95% CI 1340-1826) for those aged 13-17 years, 1395 (1175-1655) for 6-12 years, and 2406 (1942-2979) for 1-5 years. Anti-glycoprotein Ebola virus IgG antibody responses persisted up to 12 months post-vaccination, with a GMC of 716 (95% CI 619-828) for those aged 13-17 years, 752 (645-876) for 6-12 years, and 1424 (1119-1814) for 1-5 years. INTERPRETATION: ChAd3-EBO-Z was immunogenic and well tolerated in children aged 1-17 years. This study provides the first ChAd3-EBO-Z data in a paediatric population. Further development should focus on multivalent approaches including Sudan and Marburg strains, and heterologous prime-boost strategies, for instance using modified vaccinia Ankara-based vaccine to boost the immune response. FUNDING: EU's Horizon 2020 research and innovation programme and GlaxoSmithKline Biologicals SA.

Evaluation of corrective actions of feedback from clinicians on Clinical Laboratory Services at Bamenda Regional Hospital Laboratory, Cameroon

Background: Customers' satisfaction is imperative for success. Clinical laboratories continuously strive to attain very high levels of customer satisfaction to serve their clients and maintain accreditation. The concept of customer satisfaction has not yet been asserted in most clinical laboratories in Cameroon. Objectives: Our objectives were to assess the satisfaction of clinicians with the laboratory services at the Bamenda Regional Hospital Laboratory, identify important challenges, corrective actions implemented and changes in satisfaction. Methods: This retrospective study reviewed secondary data from clinician satisfaction survey records from March 2017 and November 2017. Challenges and implemented corrective actions were identified for assessed statements of dissatisfaction (dissatisfaction rates ≥ 20%) on the March 2017 survey. Satisfaction rates in March 2017 and November 2017 were compared. Results: High levels of dissatisfaction were observed for general satisfaction, waiting time, communication, duty consciousness, specimen collection and approach on the March 2017 survey. The main challenges identified were: lack of respect for the expected length of the waiting time, poor attitude, inadequate information, staff shortage and inadequate supervision. Statistically significant reductions in rates of dissatisfaction were observed for general satisfaction, waiting time, communication, response to emergencies, issuing of results, specimen collection, approach and duty consciousness. Conclusion: Waiting time is a major cause of clinician dissatisfaction with laboratory services. The identification of clinicians' challenges and the effective implementation of corrective actions contribute to improvements in clinician satisfaction

Systematic screening for tuberculosis among hospital outpatients in Cameroon: The role of screening and testing algorithms to improve case detection.

BACKGROUND: Better screening and testing approaches are needed to improve TB case finding, particularly in health facilities where many people with TB seek care but are not diagnosed using the existing approaches. OBJECTIVE: We aimed to evaluate the performance of various TB screening and testing approaches among hospital outpatients in a setting with a high prevalence of HIV/TB. METHODS: We screened outpatients at a large hospital in Cameroon using both chest X-ray and a symptom questionnaire including current cough, fever, night sweats and/or weight loss. Participants with a positive screen were tested for TB using smear microscopy, the Xpert MTB/RIF assay, and culture. RESULTS: Among 2051 people screened, 1137 (55%) reported one or more TB symptom and 389 (19%) had an abnormal chest X-ray. In total, 1255 people (61%) had a positive screen and 31 of those screened (1.5%) had bacteriologically confirmed TB. To detect TB, screening with cough >2 weeks had a sensitivity of 61% (95% CI, 44-78%). Screening for a combination of cough >2 -weeks and/or abnormal chest X-ray had a sensitivity of 81% (95% CI, 67-95%) and specificity of 71% (95% CI, 69-73%), while screening for a combination of cough >2 weeks or any of 2 or more symptoms had a similar performance. Smear microscopy and Xpert MTB/RIF detected 32% (10/31) and 55% (17/31), respectively, of people who had bacteriologically-confirmed TB. CONCLUSIONS: Screening hospital outpatients for cough >2 weeks or for at least 2 of current cough, fever, night sweats or weight loss is a feasible strategy that had a high relative yield to detect bacteriologically-confirmed TB in this population. Clinical diagnosis of TB is still an important need, even where Xpert MTB/RIF testing is available.

The evolution of HIV-1 group M genetic variability in Southern Cameroon is characterized by several emerging recombinant forms of CRF02_AG and viruses with drug resistance mutations.

The HIV epidemic in Cameroon is marked by a broad genetic diversity dominated by circulating recombinant forms (CRFs). Studies performed more than a decade ago in urban settings of Southern Cameroon revealed a dominance of the CRF02_AG and clade A variants in >90% of the infected subjects; however, little is known about the evolving viral variants circulating in this region. To document circulating HIV viral diversity, four regions of the viral genome (gag, PR, reverse transcriptase, env) in 116 HIV-1 positive individuals in Limbe, Southern Cameroon, were PCR-amplified. Sequences obtained at the RT and protease regions were analyzed for mutations that conferred drug resistance using the Stanford Drug Resistance Database. The present study reveals a broad genetic diversity characterized by several unique recombinant forms (URF) accounting for 36% of infections, 48.6% of patients infected with CRF02_AG, and the emergence of CRF22_01A1 in 7.2% of patients. Three out of 15 (20%) treated patients and 13 out of 93 (13.9%) drug naïve patients harbor drug resistance mutations to RT inhibitors, while 3.2% of drug naïve patients harbor drug resistance mutations associated with protease inhibitors. The high proportion (13.9%) of drug resistance mutations among the drug naïve patients reveals the ongoing transmission of these viruses in this region of Cameroon and highlights the need for drug resistance testing before starting treatment for patients infected with HIV-1.

Sexual behavior of HIV-positive women in Cameroon.

This study aimed at describing the sexual behavior of HIV-positive women in Cameroon. In a cross-sectional study, 282 HIV-infected women were enrolled in 3 HIV-treatment clinics in Cameroon. Of the 282 participants, 257 had been diagnosed with HIV for more than 6 months. Approximately half (46.8%) of these 257 women reported no sex partners in the 6 months before the study; 42.9% had 1 partner; and 1.5% had more than 1 partner. There was a significant decrease in the number of partners, new partners, and an increase in condom use with these partners following HIV diagnosis (P value < .05). However, more than half (55.2%) of the sexually active participants reported inconsistent or no condom use during sexual intercourse. Although HIV-positive women tend to adopt less risky behavior after HIV diagnosis, a substantial proportion of sexually active ones still have risky behaviors. Reinforcing risk reduction programs for these women is imperative.

Numbering questionnaires had no impact on the response rate and only a slight influence on the response content of a patient safety culture survey: a randomized trial.

OBJECTIVES: In self-completed surveys, anonymous questionnaires are sometimes numbered so as to avoid sending reminders to initial nonrespondents. This number may be perceived as a threat to confidentiality by some respondents, which may reduce the response rate, or cause social desirability bias. In this study, we evaluated whether using nonnumbered vs. numbered questionnaires influenced the response rate and the response content. STUDY DESIGN AND SETTING: During a patient safety culture survey, we randomized participants into two groups: one received an anonymous nonnumbered questionnaire and the other a numbered questionnaire. We compared the survey response rates and distributions of the responses for the 42-questionnaire items across the two groups. RESULTS: Response rates were similar in the two groups (nonnumbered, 75.2%; numbered, 72.8%; difference, 2.4%; P=0.28). Five of the 42 questions had statistically significant differences in distributions, but these differences were small. Unexpectedly, in all five instances, the patient safety culture ratings were more favorable in the nonnumbered group. CONCLUSION: Numbering of mailed questionnaires had no impact on the response rate. Numbering influenced significantly the response content of several items, but these differences were small and ran against the hypothesis of social desirability bias.

Retention in an antiretroviral therapy programme during an era of decreasing drug cost in Limbe, Cameroon.

BACKGROUND: In 2002, Cameroon initiated scale up of antiretroviral therapy (ART); on 1 October 2004, a substantial reduction in ART cost occurred. We assessed the impact of this event and other factors on enrolment and retention in care among HIV-infected patients initiating ART from February 2002 to December 2005 at the single ART clinic serving the Southwest Region in Limbe, Cameroon. METHODS: We retrospectively analyzed clinical and pharmacy payment records of HIV-infected patients initiating ART according to national guidelines. We compared two cohorts of patients, enrolled before and after 1 October 2004, to determine if price reduction was associated with enhanced enrolment. We assessed factors associated with retention and survival by Cox proportional hazards models. Retention in care implied patients who had contact with the healthcare system as of 31 December 2005 (including those who were transferred to continue care in other ART centres), although these patients may have interrupted therapy at some time. A patient who was not retained in care may have dropped out (lost to follow up) or died. RESULTS: Mean enrolment rates for 2920 patients who initiated ART before and after the price reduction were 46.5 and 95.5 persons/month, respectively (p < 0.001). The probabilities of remaining alive and in care were 0.66 (95% CI 0.64-0.68) at six months, 0.58 (95% CI 0.56-0.60) at one year, 0.47 (95% CI 0.45-0.49) at two years and 0.35 (95% CI 0.32-0.38) at three years; they were not significantly different between the two cohorts of patients enrolled before and after the price reduction over the first 15 months of comparable follow up (hazard ratio 1.1; 95% CI 0.9-1.2, p = 0.27). In multivariable analysis using multiple imputations to compensate for missing values, factors associated with dropping out of care or dying were male gender (HR 1.33 [1.18-1.50], p = 0.003), treatment paid by self, family or partly by other (HR 3.05 [1.99-4.67], p < 0.001), and, compared with residents of Limbe, living more than 150 km from Limbe (HR 1.41 [1.18-1.69], p < 0.001), or being residents of Douala (HR 1.51 [1.16-1.98], p < 0.001). CONCLUSIONS: Reducing the cost of ART increased enrolment of clients in the programme, but did not change retention in care. In a system where most clients pay for ART, an accessible clinic location may be more important than the cost of medication for retention in care. Decentralizing ART clinics might improve retention and survival among patients on ART.

Sequence analysis of the dimerization initiation site of concordant and discordant viral variants superinfecting HIV type 1 patients.

For HIV recombination to occur, the RNAs from two infecting strains within a cell must dimerize at the dimerization initiation site (DIS). We examined the sequence identity at the DIS (697-731 bp, Hxb2 numbering engine) in patients superinfected with concordant HIV-1 strains and compared them to those with discordant strains. Viral RNA in sequential plasma from four subjects superinfected with subtype-discordant and two subjects superinfected with subtype-concordant HIV-1 strains was extracted, amplified (5' LTR-early gag: 526-1200 bp, Hxb2 numbering engine), sequenced, and analyzed to determine their compatibility for dimerization in vivo. The concordant viruses infecting the two subjects exhibited identical sequences in the 35-bp-long DIS region while sequences from the discordant viruses revealed single nucleotide changes that were located in the DIS loop (715 bp), its flanking nucleotides (710 bp and 717 bp), and the DIS stem (719 bp). Evidence from in vitro experiments demonstrates that these in vivo changes identified can abolish dimerization and reduce recombination frequency. Therefore, these results revealing differences in the DIS of discordant strains versus the similarity noted for the concordant strains may contribute to the differences in the frequency of recombination in patients superinfected with such HIV-1 variants.

Infection by discordant strains of HIV-1 markedly enhances the neutralizing antibody response against heterologous virus.

High-risk cohorts in East Africa and the United States show rates of dual HIV-1 infection--the concomitant or sequential infection by two HIV-1 strains--of 50% to 100% of those of primary infection, and our normal-risk HIV-positive cohort in Cameroon exhibits a rate of dual infection of 11% per year, signifying that these infections are not exceptional. Little is known regarding the effect of dual infections on host immunity, despite the fact that they provide unique opportunities to investigate how the immune response is affected when challenged with diverse HIV-1 antigens. Using heterologous primary isolates, we have shown here that dual HIV-1 infection by genetically distant strains correlates with significantly increased potency and breadth of the anti-HIV-1 neutralizing antibody response. When the neutralization capacities of sequential plasma obtained before and after the dual infection of 4 subjects were compared to those of matched plasma obtained from 23 singly infected control subjects, a significant increase in the neutralization capacity of the sequential sample was found for 16/28 dually infected plasma/virus pairs, while only 4/159 such combinations for the control subjects exhibited a significant increase (P < 0.0001). Similarly, there was a significant increase in the plasma dilution capable of neutralizing 50% of virus (IC(50)) for 18/24 dually infected plasma/virus pairs, while 0/36 controls exhibited such an increase (P < 0.0001). These results demonstrate that dual HIV-1 infection broadens and strengthens the anti-HIV-1 immune response, suggesting that vaccination schemes that include polyvalent, genetically divergent immunogens may generate highly protective immunity against any HIV-1 challenge strain.

Longitudinal quasispecies analysis of viral variants in HIV type 1 dually infected individuals highlights the importance of sequence identity in viral recombination.

Little is known regarding the likelihood of recombination between any given pair of nonidentical HIV-1 viruses in vivo. The present study analyzes the HIV-1 quasispecies in the C1C2 region of env, the vif-vpr-vpu accessory gene region, and the reverse transcriptase region of pol. These sequences were amplified from samples obtained sequentially over a 12- to 33-month period from five dually HIV-1-infected subjects. Analysis of an average of 248 clones amplified from each subject revealed no recombinants within the three loci studied of the subtype-discordant infecting strains, whose genetic diversity was >11% in env. In contrast, two subjects who were initially coinfected by two subtype-concordant variants with genetic diversity of 7.4% in env were found to harbor 10 unique recombinants of these strains, as exhibited by analysis of the env gene. The frequent recombination observed among the subtype-concordant strains studied herein correlates with prior sequence analyses that have commonly found higher rates of recombination at loci bearing the most conserved sequences, demonstrating an important role for sequence identity in HIV-1 recombination. Viral load analysis revealed that the samples studied contained an average of 8125 virus copies/ml (range, 882-31,626 copies/ml), signifying that the amount of viral RNA in the samples was not limiting for studying virus diversity. These data reveal that recombination between genetically distant strains may not be an immediate or common outcome to dual infection in vivo and suggest critical roles for viral and host factors such as viral fitness, virus diversity, and host immune responses that may contribute to limiting the frequency of intersubtype recombination during in vivo dual infection.

HIV-1 reverse transcriptase drug-resistance mutations in chronically infected individuals receiving or naïve to HAART in Cameroon.

The most common first-line, highly active anti-retroviral therapy (HAART) received by individuals infected with HIV-1 in Cameroon is the combination therapy Triomune, comprised of two nucleoside reverse transcriptase inhibitors (NRTI) and one non-NRTI (NNRTI). To examine the efficacy of these drugs in Cameroon, where diverse non-B HIV-1 subtypes and recombinant viruses predominate, the reverse transcriptase (RT) viral sequences in patient plasma were analyzed for the presence of mutations that confer drug resistance. Forty-nine HIV-1-positive individuals were randomly selected from those receiving care in HIV/AIDS outpatient clinics in the South-West and North-West Regions of Cameroon. Among the 28 patients receiving HAART, 39% (11/28) had resistance to NRTIs, and 46% (13/28) to NNRTIs after a median of 12 months from the start of therapy. Among those with drug-resistance mutations, there was a median of 14 months from the start of HAART, versus 9 months for those without; no difference was observed in the average viral load (10,997 copies/ml vs. 8,056 copies/ml). In contrast, drug-naïve individuals had a significantly higher average viral load (27,929 copies/ml) than those receiving HAART (9,527 copies/ml). Strikingly, among the 21 drug-naïve individuals, 24% harbored viruses with drug-resistance mutations, suggesting that HIV-1 drug-resistant variants are being transmitted in Cameroon. Given the high frequency of resistance mutations among those on first-line HAART, coupled with the high prevalence of HIV-1 variants with drug-resistance mutations among drug-naïve individuals, this study emphasizes the need for extensive monitoring of resistance mutations and the introduction of a second-line HAART strategy in Cameroon.

High frequency of HIV-1 dual infections among HIV-positive individuals in Cameroon, West Central Africa.

OBJECTIVES: To determine the frequency of dual inter- and intra-subtype HIV-1 infection among a cohort of 64 longitudinally-studied, HIV-1-positive individuals in Yaoundé, Cameroon. METHODS: Blood was collected every 3-6 months for up to 36 months and RNA was extracted from plasma. Gag fragment (HxB2 location 1577-2040) was amplified by nested RT-PCR, and mixed-time-point Heteroduplex Assays (HDAs) were performed. As heteroduplexes in this assay indicate >or=5% genetic discordance in the gag fragment, their presence reveals dual infection. Results were confirmed by phylogenetic analysis. RESULTS: Heteroduplexes were generated by specimens of 10 subjects (15.6%). Kaplan-Meier nonparametric estimate of maintenance of single infection was calculated; the rate/year of a 2 infection was found to be approximately 11%. Dual infection was identified in the final specimens of five subjects, after as much as 18 months follow-up, while for the remaining five subjects, dual infection was identified in interim specimens within an average of 10 months follow-up. Analysis of samples obtained after dual infection from each of these latter five subjects revealed two patterns: reversion to initial strain, or replacement of initial strain. Four subjects were dually-infected with HIV-1 strains of the same subtype, while 6 were infected with different subtypes. CONCLUSIONS: The high prevalence of recombinant HIV-1 strains in Cameroon may in part be explained by the high frequency of dual infection. In this genetically-diverse HIV-1 milieu, dual infections and the recombinant viruses they generate are strongly driving viral evolution, complicating vaccine strategies.

Utility of the heteroduplex assay (HDA) as a simple and cost-effective tool for the identification of HIV type 1 dual infections in resource-limited settings.

The predominance of unique recombinant forms (URFs) of HIV-1 in Cameroon suggests that dual infection, the concomitant or sequential infection with genetically distinct HIV-1 strains, occurs frequently in this region; yet, identifying dual infection among large HIV cohorts in local, resource-limited settings is uncommon, since this generally relies on labor-intensive and costly sequencing methods. Consequently, there is a need to develop an effective, cost-efficient method appropriate to the developing world to identify these infections. In the present study, the heteroduplex assay (HDA) was used to verify dual or single infection status, as shown by traditional sequence analysis, for 15 longitudinally sampled study subjects from Cameroon. Heteroduplex formation, indicative of a dual infection, was identified for all five study subjects shown by sequence analysis to be dually infected. Conversely, heteroduplex formation was not detectable for all 10 HDA reactions of the singly infected study subjects. These results suggest that the HDA is a simple yet powerful and inexpensive tool for the detection of both intersubtype and intrasubtype dual infections, and that the HDA harbors significant potential for reliable, high-throughput screening for dual infection. As these infections and the recombinants they generate facilitate leaps in HIV-1 evolution, and may present major challenges for treatment and vaccine design, this assay will be critical for monitoring the continuing pandemic in regions of the world where HIV-1 viral diversity is broad.