Chikungunya in the western hemisphere: a review of the 2014 epidemic, the potential long-term impact, and research opportunities

OBJECTIVE: The objective of this paper is to present a comprehensive review of the impact of the recent chikungunya virus outbreaks in island nations, including the Caribbean, and explore the potential for further study of the epidemiology and pathogen-host interactions of this emerging virus. DESIGN AND METHODS: We conducted a review of the current literature and data on multiple facets of chikungunya including: acute disease outbreaks, epidemiological and clinical data, comparisons of diagnostic techniques, and virologic strains. RESULTS: Outbreaks of chikungunya (CHIKV) on island nations have seen high attack rates with corresponding increased morbidity and mortality. Severe, persistent and relapsing arthritis and tenosynovitis is common among chikungunya patients. CONCLUSION: Comprehensive surveillance of chikungunya virus is required by the linking of epidemiologic, molecular and immunologic data with information on ecological patterns and vector prevalence if the incidence of chikungunya is to be reduced and prevented. More data on the disease spectrum and persistence in the Caribbean nations, the viral strain, and the incidence rates are sorely needed. Because CHIK can only be prevented by preventing mosquito bites, more research needs to be done locally in Caribbean nations to determine the optimal strategies of Aedes vector control and public health education with subsequent behavior change.

Clinical, molecular and serological outcomes of the Chikungunya outbreak in Grenada

OBJECTIVE: This study evaluated the clinical utility of Chikungunya (CHIKV) test results and clinical symptoms in patients with suspected CHIKV infection. DESIGN AND METHODS: Patients with CHIKV symptoms who presented at a health facility in Grenada during the recent outbreak had a CHIKV diagnostic test form completed by a health professional and a blood sample was drawn. The serum sample was stored at -80oC, shipped to the Naval Infectious Diseases Diagnostic Lab (NIDDL) on dry ice and tested for CHIKV and Dengue (DENV) using PCR real-time assay for viral RNA, and IgM detection by ELISA. RESULTS: Sera from more than 600 patients collected from mid September till mid October, 2014 were drawn and had a CHIKV diagnostic form completed. At the time of writing 112 patients sera have been tested at the NIDDL. 90% of patients had a positive test. PCR only was positive in 8% of patients. IgM only was positive in 83%, and both PCR and IgM were positive in 9% of patients. The major symptoms presented by patients were joint pain (84%), fever (81%), body pain (74%), headache (62%), chills (54%) and rash (49%). CONCLUSION: IgM testing detected 92% of test positive patients while PCR alone detected 17%. The IgM assay was clinically most useful. In an outbreak where dengue is ruled out and CHIKV is the cause, patients with the constellation of symptoms above could be considered positive for CHIKV infection with a 98% accuracy without confirmatory testing.

Lessons learnt from the intense 2014 Chikungunya Epidemic in Grenada

OBJECTIVE: To present an overview of the lessons learnt from the fall 2014 outbreak of Chikungunya (CHIK) in Grenada. DESIGN AND METHODS: A review of newspaper articles, news reports and opinions of clinicians and policy makers on the impact and evolution of the CHIK outbreak in Grenada was conducted. RESULTS: CHIK outbreaks on small island developing nations are characterized by high attack rates. The speed of the spread of the virus is facilitated by the efficient domesticated diurnal vector species Aedes aegypti. Efforts to educate the public and to control this vector stretched resources. Clinical attack rates in the Grenada outbreak impacted manpower resources in every sector including clinical services. CONCLUSION: CHILK infected an estimated 60% of the population in just three months of intense transmission. The resulting morbidity meant that essential lessons were learnt. These included the need for a rapid response in educating the population on the mode of transmission of the virus and its prevention, the implementation of vector control and the demand for diagnostic tests. Essential services were short staffed. The need for an unprecedented rapid response and the impact of the CHIK outbreak in Grenada will be presented.

Establishment of laboratory testing capability for chikungunya virus in Grenada, West Indies

OBJECTIVE: Rapid diagnosis of Chikungunya (CHIKV) is important early in an epidemic. The study objective was to describe the process of implementing CHIKV testing capability in Grenada and to confirm the arrival of CHIKV on the main island of Grenada. DESIGN AND METHODS: In April, 2014 a collaborative study between the U.S. Naval Infectious Diseases Diagnostic Laboratory (NIDDL) and the clinical microbiology laboratory of St. George’s University (SGU) was started. SGU acquired essential instrumentation and patient samples, and NIDDL provided supplies and reagents, plus technical training experts. RESULTS: Personnel, supplies and equipment arrived in Grenada in August 2014. Set up of instruments and test validation were completed quickly. Initial CHIKV PCR and IgM tests found 3 PCR positive samples. The IgM assay found several presumptive positives that were unable to be confirmed due to ELISA instrument malfunction. PCR data indicated that CHIKV had arrived on the main island of Grenada no later than August, 2014. Based on arbovirus test demand, symptomatic patients began to increase in August, peaked in September, and tailed off during November. CONCLUSION: Both CHIKV tests were implemented and produced the first on-island reference test confirmation of CHIKV patients in Grenada. The most difficult part of this effort was training technologists in time to help with testing. Laboratory testing for CHIKV infection can be a challenge in developing states at a distance from support services. Collaborative links with established labs remains essential.

Fatal case of Weeksella virosa sepsis.

Weeksella virosa is an aerobic Gram-negative rod that has rarely been reported to cause infection. We describe a fatal case of W. virosa sepsis in a young female with end-stage renal disease, report three additional cases of W. virosa infection, and review the literature regarding this infection.

Emergence of staphylococcal cassette chromosome mec type IV methicillin-resistant Staphylococcus aureus as a cause of ventilator-associated pneumonia.

Staphylococcal cassette chromosome mec (SCCmec) type IV methicillin-resistant Staphylococcus aureus (MRSA) strains were identified in 8 (19.5%) of 41 consecutive patients with MRSA ventilator-associated pneumonia (VAP) in this retrospective, observational study. There were no significant differences in VAP severity and crude mortality rates between patients with SCCmec type II strains and patients with SCCmec type IV strains.

Enhancing the specificity of the COBAS AMPLICOR CT/NG test for Neisseria gonorrhoeae by retesting specimens with equivocal results.

The COBAS AMPLICOR CT/NG test for Neisseria gonorrhoeae cross-reacts with certain strains of nonpathogenic Neisseria species. In some strains, the target sequence is identical to that of N. gonorrhoeae, whereas other strains have a small number of mismatches within the regions recognized by the primers or probe used in the COBAS AMPLICOR NG test. These cross-reactive strains are occasionally present in urogenital specimens, causing false-positive results in the COBAS AMPLICOR NG test. Analysis of the data generated in a large multicenter clinical trial showed that 2.9% of the specimens gave signals between A(660)s of 0.2 and 3.5 but that one-half of these equivocal specimens did not contain N. gonorrhoeae. Most of these equivocal specimens were correctly classified as true positive or true negative by retesting in duplicate and defining a PCR-positive result as two of three results with an A(660) of > or =2.0. If specimens had been classified as positive or negative based on a single test result using a cutoff of an A(660) of 0.2, specificity would have ranged from 96.2 to 98.9% depending on specimen type, sex, and presence of symptoms. By employing the equivocal zone-retesting algorithm, specificity increased to 98.6 to 99.9% with little effect (0.1 to 4.9% decrease) on sensitivity in most specimen types, enabling the test to achieve a positive predictive value of at least 90% in populations with a prevalence of 4% or higher. In lower-prevalence populations, the test could be used to screen for presumptive infections that would have to be confirmed by an independent test.

Automation of laboratory testing for infectious diseases using the polymerase chain reaction-- our past, our present, our future.

While it is an extremely powerful and versatile assay method, polymerase chain reaction (PCR) can be a labor-intensive process. Since the advent of commercial test kits from Roche and the semi-automated microwell Amplicor system, PCR has become an increasingly useful and widespread clinical tool. However, more widespread acceptance of molecular testing will depend upon automation that allows molecular assays to enter the routine clinical laboratory. The forces driving the need for automated PCR are the requirements for diagnosis and treatment of chronic viral diseases, economic pressures to develop more automated and less expensive test procedures similar to those in the clinical chemistry laboratories, and a shortage in many areas of qualified laboratory personnel trained in the types of manual procedures used in past decades. The automated Roche COBAS AMPLICOR system has automated the amplification and detection process. Specimen preparation remains the most labor-intensive part of the PCR testing process, accounting for the majority of the hands-on-time in most of the assays. A new automated specimen preparation system, the COBAS AmpliPrep, was evaluated. The system automatically releases the target nucleic acid, captures the target with specific oligonucleotide probes, which become attached to magnetic beads via a biotin-streptavidin binding reaction. Once attached to the beads, the target is purified and concentrated automatically. Results of 298 qualitative and 57 quantitative samples representing a wide range of virus concentrations analyzed after the COBAS AmpliPrep and manual specimen preparation methods, showed that there was no significant difference in qualitative or quantitative hepatitis C virus (HCV) assay performance, respectively. The AmpliPrep instrument decreased the time required to prepare serum or plasma samples for HCV PCR to under 1 min per sample. This was a decrease of 76% compared to the manual specimen preparation method. Systems that can analyze more samples with higher throughput and that can answer more questions about the nature of the microbes that we can presently only detect and quantitate will be needed in the future.

Multicenter evaluation of AMPLICOR and automated COBAS AMPLICOR CT/NG tests for Neisseria gonorrhoeae.

The fully automated COBAS AMPLICOR CT/NG and semiautomated AMPLICOR CT/NG tests were evaluated in a multicenter trial for their ability to detect Neisseria gonorrhoeae infections. Test performance compared to that of culturing was evaluated for 2,192 matched endocervical swab and urine specimens obtained from women and for 1, 981 matched urethral swab and urine specimens obtained from men. Culture-negative, PCR-positive specimens that tested positive in a confirmatory PCR test for an alternative target sequence within the N. gonorrhoeae 16S rRNA gene were considered to be true positives. The overall prevalences of gonorrhea were 6.6% in women and 20.1% in men. The COBAS AMPLICOR and AMPLICOR formats yielded concordant results for 98.8% of the specimens and exhibited virtually identical sensitivities and specificities. The results that follow are for the COBAS AMPLICOR format. With the infected patient as the reference standard, the resolved sensitivities of PCR were 92.4% for endocervical swab specimens and 64.8% for female urine specimens. There were no significant differences in these rates between women with and without symptoms. Among symptomatic men, COBAS AMPLICOR sensitivities were 94.1% for urine and 98.1% for urethral swabs; for asymptomatic men, the results were 42.3 and 73.1%, respectively. In comparison, the sensitivities of culturing were 84.8% for endocervical specimens, 92.7% for symptomatic male urethral specimens, and only 46.2% for urethral specimens obtained from asymptomatic men. When PCR results were analyzed as if only a single test had been performed on a single specimen type, the resolved sensitivity was always higher. The resolved specificities of PCR were 99.5% for endocervical swab specimens, 99.8% for female urine specimens, 98.9% for male urethral swab specimens, and 99.9% for male urine specimens. The internal control revealed that 2.1% of specimens were inhibitory when initially tested. Nevertheless, valid results were obtained for 99.2% of specimens because 60.0% of the inhibitory specimens were not inhibitory when a second aliquot was tested. The COBAS AMPLICOR CT/NG test for N. gonorrhoeae exhibited high sensitivity and specificity with urethral swab and urine specimens from men and endocervical swab specimens from women and thus is well suited for diagnosing and screening for N. gonorrhoeae infection.

Multicenter evaluation of the AMPLICOR and automated COBAS AMPLICOR CT/NG tests for detection of Chlamydia trachomatis.

The fully automated COBAS AMPLICOR CT/NG and semiautomated AMPLICOR CT/NG tests were evaluated in a multicenter trial for the ability to detect Chlamydia trachomatis infections. Test performance compared to that of culture was evaluated for 2,236 matched endocervical swab and urine specimens obtained from women and for 1,940 matched urethral swab and urine specimens obtained from men. Culture-negative, PCR-positive specimens that tested positive in a direct fluorescent-antibody test or in a confirmatory PCR test for an alternative target sequence were resolved as true positives. The overall prevalences of chlamydia were 2.4% in women and 7.2% in men. The COBAS AMPLICOR and AMPLICOR formats yielded concordant results for 98.1% of the specimens. With the infected patient as the reference standard, the resolved sensitivities of COBAS AMPLICOR were 89.7% for endocervical swab specimens, 89.2% for female urine specimens, 88.6% for male urethral swab specimens, and 90.3% for male urine specimens. When results were analyzed as if only a single test had been performed on a single specimen type, the resolved sensitivity was always higher. The resolved specificities of PCR were 99.4% for endocervical swab specimens, 99.0% for female urine specimens, 98.7% for male urethral swab specimens, and 98.4% for male urine specimens. The internal control revealed that 2.4% of the specimens were inhibitory when initially tested. Nevertheless, valid results were obtained for 98.6% of the specimens because 59.1% of the inhibitory specimens were not inhibitory when a second aliquot was tested. The COBAS AMPLICOR and AMPLICOR CT/NG tests for C. trachomatis exhibited equally high sensitivity and specificity with both urogenital swab and urine specimens and thus are well suited for screening for C. trachomatis infection.

A receptor-specific peptide for imaging infection and inflammation.

The chemotactic peptide N-formylmethionyl-leucyl-phenylalanine (fMLP), when radiolabelled, continues to be an attractive agent for imaging infection or inflammation. Previously, several analogues of fMLP have been prepared and radiolabelled using a bifunctional chelating agent conjugation procedure that was relatively long and complex. We have prepared a new analogue of fMLP, TP765, by the addition of 4-aminobutyric acid (4-ABA) and a group of four amino acids, Gly-Gly-d-Ala-Gly, to the carboxy terminus (i.e. to the phenylalanine) of fMLP. The adduct -(4-ABA)-Gly-Gly-d-Ala-Gly- serves as a chelating moiety for strong chelation with 99Tcm. The use of a peptide as a chelating moiety greatly simplified the synthetic procedure and rendered the analogue ready for instant chelation with 99Tcm. HPLC analysis revealed that 99Tcm-TP765 was a single chemical entity that retained biological activity and neutrophil specificity. 99Tcm-TP765 was stable when challenged with strong chelating agents in vitro and had rapid but biphasic blood clearance (alphat1/2 = 7 min, betat1/2 = 45 min). Approximately 90% of the radioactivity had cleared from circulation within 45 min post-injection and the agent had accumulated in experimental bacterial or sterile abscesses in significantly (P<0.05) higher quantities than the analogues evaluated previously. Generally, the biodistribution pattern of 99Tcm-TP765 was similar to that of other analogues examined and its abscess uptake was independent of the abscess age. In conclusion, a new analogue of fMLP, 99Tcm-TP765, was prepared by a simple procedure. This new analogue has properties similar to those of previously examined analogues used as agents for imaging infection or inflammation.

Evaluation of AMPLILINK software for the COBAS AMPLICOR system.

The use of AMPLILINK version 1.0 software was evaluated for the operation and control of one COBAS AMPLICOR instrument and for two COBAS AMPLICOR instruments run simultaneously to perform and detect nucleic acid amplification reactions. A total of 3,384 results were analyzed. The initial accuracy of the results was 99.91%. Three errors of omission of transfer of data from the COBAS AMPLICOR to the AMPLILINK system were observed. Two of these errors were from a single specimen, where both the analyte and internal control results were not transmitted. These errors did not interfere with the correctness of any other data. There were no interruptions of runs, and no data were mixed. AMPLILINK increased convenience, saved labor, and was found to be a very useful addition for clinical laboratories performing molecular-diagnostic procedures with the COBAS AMPLICOR system.

Some characteristics of platelet concentrates contaminated with Staphylococcus epidermidis.

The study investigates platelet concentrates (PCs) contaminated with Staphylococcus epidermidis. The work was conducted using 12 pairs of PCs. Light transmission through the PCs was observed over 7 days. Immediately after preparation, one PC was inoculated with Staphylococcus epidermidis (17-43 colony forming units (CFU)/ ml) and the other served as a non-contaminated control. In the contaminated PCs the CFU/ml and swirling were determined on days 1, 3 and 7. In all PCs determination of pH and the extent of shape change (ESC) was carried out on days 3 and 7. All contaminated PCs had visible swirling on day 3 but they contained 2.0 X 10 4 to 9.0 X 10 8 CFU/ml. pH (P=0.05) and ESC (P<0.05) then were slightly lower. On day 7, eight of 12 of the contaminated PCs failed to display transmission changes despite deranged analytical results and impaired swirling. During early storage the contaminated units had unimpaired swirling and only slightly impaired in vitro quality. They appear to be difficult to identify in the routine.

Viral burden and disease progression in HIV-1-infected patients with sickle cell anemia.

The spleen and lymph nodes are major sites of human immunodeficiency virus type 1 (HIV-1) replication, mutation, and genetic variation in vivo. If a major portion of the lymphatic tissue, such as the spleen, is removed or otherwise is unavailable for invasion by the HIV-1 virus, will the course of the infection be altered, resulting in a prolonged symptom-free interval or even increased survival? The spleen of most adults with sickle cell anemia (SS) is nonfunctional due to recurrent episodes of microinfarction. If autosplenectomized SS patients are exposed to HIV-1, they may be ideal candidates to examine the question of whether absence of splenic function at the time of infection will positively alter the course of HIV-1-related disease. All SS patients with a diagnosis of HIV-1 infection at five university sickle cell centers were included in the patient cohort. Patients in active treatment or in follow-up (group A, n = 11) underwent a series of quantitative viral studies to determine their HIV-1 viral burden. The studies included the branched-DNA signal amplification assay, quantitative DNA-polymerase chain reaction (PCR), quantitative reverse transcription (RT)-initiated-PCR, and in situ PCR. All patients who died of the complications of the acquired immunodeficiency syndrome (AIDS) or of SS, lost to follow-up, or were otherwise unavailable for study (Group B: n = 7) were included in the total patient group. None of the patients in group B underwent quantitative viral studies. In addition, a control population (group C, n = 36) of HIV-1-infected African Americans without SS, of similar age and gender to the SS patients, were compared with the study population for outcomes. In eight of 11 active patients (group A), the CD4+ T-lymphocyte counts were normal and viral burdens were low for an average of 10.25 years following diagnosis. These eight patients all from group A were the only long-term nonprogressors (44%) among a total of 18 SS patients (groups A and B). In group C (control), only five patients of 36 were long-term nonprogressors (13.9%). Five patients (28%) of the total SS group (groups A and B) succumbed to AIDS. One of the five was from Group A. The evaluation of a limited number of adult individuals suggests that a significant proportion of HIV-1-seropositive SS patients (44%) may be asymptomatic long-term nonprogressors. In these patients, the CD4+ T-lymphocyte counts remained high and their viral burdens were remarkably lower than in non-SS HIV-1-seropositive individuals. Whereas this study does not prove an "autosplenectomy" hypothesis, it suggests that in patients with both SS and HIV-1 infection, the retroviral disease may be ameliorated by host factors of which absence of splenic function prior to HIV-1 infection may be one.

Confirmation of the Syva MicroTrak enzyme immunoassay for chlamydia trachomatis by Syva Direct Fluorescent Antibody Test.

BACKGROUND AND OBJECTIVES: The Syva Micro Trak enzyme immunoassay (EIA) is used widely for screening women infected with Chlamydia trachomatis. Confirmatory tests used in conjunction with EIA screening have shown that false-positive results are common. GOALS: To evaluate the specificity of the Syva MicroTrak EIA by confirmation of positive specimens with the Syva Direct Fluorescent Specimen Test. STUDY DESIGN: Of 6,039 endocervical specimens collected from women attending Colorado family planning clinics, 328 positive EIA results (5.4%) were obtained by Syva MicroTrak EIA. A random subset of 136 positive specimens was tested by Syva Direct Specimen Test. Twenty of 136 specimens (14.7%) negative by Syva Direct Specimen testing were also tested by Syva blocking antibody tests (9 of 20 positive, 45%) and Roche Amplicor polymerase chain reaction (PCR; 6 of 20 positive, 30%). Of 20 specimens positive by Syva MicroTrak EIA and negative by Syva Direct Specimen Test, 11 (55%) were also negative by blocking antibody and PCR, including three specimens with initial EIA sample-to-cutoff ratios greater than 2. CONCLUSIONS: Confirmatory testing of Syva MicroTrak EIA positive specimens with Syva Direct Specimen Test showed that 14.7% were false positive. Coupling the Syva Direct Specimen test with either blocking antibody or PCR reduces the rate of false-positive results to 8%.

Evaluation of automated COBAS AMPLICOR PCR system for detection of several infectious agents and its impact on laboratory management.

We evaluated the COBAS AMPLICOR (CA) PCR system (Roche Diagnostic Systems) designed for automated PCR amplification and detection of nucleic acids from infectious agents in clinical samples. The Roche AMPLICOR microwell plate (MWP) PCR was the reference method. CA amplifies target nucleic acid, captures the biotinylated amplification products by using magnetic particles coated with specific oligonucleotide probes, and detects the bound products colorimetrically. For Mycobacterium tuberculosis, the correlation of the results of CA tests with those of MWP tests was 100% with 230 samples, including 20 culture-positive samples. For hepatitis C virus, the correlation was 100% with 214 samples, including 60 positive samples. MultiPlex CA analysis of 199 cervical specimens for Chlamydia trachomatis, Neisseria gonorrhoeae, and the internal control gave 100% concordance. These samples included 19 C. trachomatis and 3 N. gonorrhoeae culture-positive samples. Overall, the agreement between PCR methods for all 842 comparisons was 100%. Compared with culture, the sensitivities of the assays for C. trachomatis and M tuberculosis were > or = 95%. After spiking alternating amplification tubes in the CA system with 10(14) copies of the Chlamydia amplicon per ml, we were unable to demonstrate any carryover cross-contamination of negative samples. Using the criteria of the College of American Pathologists workload recording method, we found that the total hands-on time to produce CA PCR results was 4.4, 7.9, and 3.3 min for M. tuberculosis, hepatis C virus, and the MultiPlexed assay for chlamydia plus gonorrhea and an internal control, respectively. The CA system brings true PCR automation to laboratories. In addition to the accuracy of automated results, the CA system provides labor savings, provides containment of the amplification and detection components of PCR, and supports both MultiPlex amplification and sequential algorithm (ReFlex) detection of analytes.

Evaluation of Amplicor PCR for direct detection of Mycobacterium tuberculosis from sputum specimens.

We evaluated the Amplicor PCR assay (Roche Molecular Systems, Branchburg, N.J.) for direct detection of Mycobacterium tuberculosis in sputum. A total of 532 specimens from 270 patients were decontaminated and stored at 4 or -75 degrees C until assayed by PCR. This assay used three-step sample preparation, biotinylated primer pairs, AmpErase, and a microtiter format for amplicon capture and detection. Amplicor PCR results were compared with clinical history, culture from a Lowenstein-Jensen slant, and results from the BACTEC TB-460 system. Eighty-seven cultures from 15 patients grew M. tuberculosis; of these, 83 (95%) were positive with the Amplicor PCR test. The false negatives were most likely due to sample variation and inhibitors. Of the 445 specimens from which M. tuberculosis was not isolated, 428 (96%) were negative with the Amplicor PCR test. Of the 17 M. tuberculosis culture-negative, Amplicor-positive specimens, 15 were reclassified as true positives because previous cultures grew M. tuberculosis. Of the 445 specimens which did not grow M. tuberculosis, Mycobacterium spp. other than M. tuberculosis were isolated from 150 specimens. Three of these 150 specimens were Amplicor positive; two were from a patient with a history of tuberculosis, and one specimen gave a false-positive result. We do not feel that this represents cross-reactivity, because repeated Amplicor testing of the isolate gave negative results. The microtiter plate has 96 wells. Allowing for six controls, 90 decontaminated specimens can be tested by one technologist in 7.5 h. This PCR assay took 7.5 h to complete and is a sensitive and specific, rapid method for the direct detection of M. tuberculosis from sputum.