Our findings suggest that methylene blue-induced neuroprotection is mediated, at least in part, by macroautophagy though activation of AMPK signaling.Methylene blue has been shown to be neuroprotective in multiple experimental neurodegenerative disease models. However, the mechanisms underlying the neuroprotective effects have not been fully elucidated. Previous studies have shown that macroautophagy has multiple beneficial roles for maintaining normal cellular homeostasis and that induction of macroautophagy after myocardial ischemia is protective. In the present study we demonstrated that methylene blue could protect HT22 hippocampal cell death induced by serum deprivation, companied by induction of macroautophagy. We also found that methylene blue-mediated neuroprotection was abolished by macroautophagy inhibition. Interestingly, 5′ adenosine monophosphate-activated protein kinase (AMPK) signaling, but not inhibition of mammalian target of rapamycin signaling, was activated at 12 and 24 h after methylene blue treatment in a dose-dependent manner. Methylene blue-induced macroautophagy was blocked by AMPK inhibitor. Consistent with  Methylene blue has long been used as treatment for methemoglobinemia , ifosfamMacroautophagy is one of the main mechanisms for maintaining cellular homeostasis, including for maintaining homeostasis and functions of central nervous system . In the In this study, we evaluated the involvement of macroautophagy in the methylene blue-mediated neuroprotection. Our aim was to elucidate the mechanisms by which methylene blue protects neurons during nutrient insufficiency.n = 4 in each group) were intragastrically administrated 50 (low dose) or 100mg/kg (high dose) methylene blue  daily for two consecutive weeks. Then, mice were euthanized and brains collected. Cortex, hippocampus and subventricular zone (SVZ) were isolated, snip frozen in liquid nitrogen and stored in –80°C for further analysis.C57BL/6 mice  were purchased from the Jackson Laboratory. All animal procedures were approved by the University of North Texas Health Science Center Animal Care and Use Committee. All experiments were conducted in compliance with institutional guidelines and NIH Guidelines for the Use of Animals in Neuroscience and Behavioral research. Twelve mice (2  at 37°C. Cells at passage 8~12 were adjusted to 1.5×105/ml and were plated in 12-well or 6-well cell culture plates (Corning). At 4 to 6 h after seeding, adherent cells were rinsed with phosphate buffered saline (PBS) twice and serum-free DMEM in the presence or absence of methylene blue was added for indicated time periods of culture. For methylene blue pretreatment and cell viability assay, cells were seeded into 96-well plates and cultured for 24 h in methylene blue-containing DMEM supplemented with 10% fetal calf serum, then followed by 12-h culture in serum-free DMEM. Cell viability was determined using Calcein-AM (Invitrogen) assay according to the manufacture’s instruction.Mouse HT22 hippocampal cell line was cultured in Dulbecco’s Modified Eagle Medium (DMEM) (Gibco) supplemented with 10% fetal calf serum (Hyclone), 50IU/ml penicillin and 50 μg/ml streptomycin (Invitrogen) in a humidified incubator with 5% COFloating cells and adherent cells were collected by gently flushing the wells with 2 mM EDTA-PBS. Cells were then centrifuged and stained with propidium iodide  and Annexin V (BD Biosciences) according to the manufacture’s instruction.TM chamber slides (Thermo Scientific) followed by methylene blue treatment as described above. Cells were then fixed with 4% paraformaldehyde and stained with a rabbit anti-LC3B antibody  followed by staining with Alexa Fluor 594-conjugated goat anti rabbit IgG (Invitrogen). Fluorescent microscopy was conducted using an Axio Observer Z1 fluorescent microscope (Zeiss).Cells were seeded in each well of 8-well Lab-TekTM Precast Gels (Bio-Rad). Proteins were then transferred onto nitrocellulose membranes and blocked with 5% non-fat dry milk-PBS for 1 h at room temperature (RT). After three washes with 0.05% Tween-20-PBS, membranes were incubated with the following primary antibody reagents overnight at 4°C, respectively: (1) Rabbit monoclonal anti-AMPK, anti-Phospho-AMPK, anti-ACC and anti-Phospho-ACC ; (2) Rabbit monoclonal anti-mTOR and anti-Phospho-mTOR ; (3) Rabbit monoclonal anti-Erk1/2  and anti-Phospho-Erk1/2 Thr202/Tyr204 ; (4) mouse monoclonal anti-Bcl-2 (Santa Cruz); (5) rabbit polyclonal anti-Akt (Santa Cruz) and anti-Phospho-Akt1 , and (6) mouse monoclonal anti-β-actin (Santa Cruz). Membranes were washed three times with PBST and then incubated with horseradish peroxidase-conjugated secondary antibodies for 1 h at RT. Membranes were developed with SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific) and were visualized and the optical density of the identified protein bands on membranes was analyzed using a Biospectrum 500 imaging system .Cell lysate and mouse brain tissue lysate were prepared by homogenization in RIPA buffer  and lysis on ice for 30 min. Protein amount was quantified using Pierce 660 nm Protein Assay (Thermo Scientific) following the manufacture’s instruction. Total protein from each sample was loaded onto 8% (for acetyl-CoA carboxylase (ACC) and mammalian target of rapamycin (mTOR) or 10% , Akt, 5′ adenosine monophosphate-activated protein kinases (AMPKs), B-cell lymphoma 2 (Bcl-2), and β-actin) SDS-PAGE gel and electrophoresis was performed. For LC3B, proteins were loaded onto 4–20% Mini-PROTEAN® TGXt test or one-way ANOVA was used for comparison of mean between the groups, and p < 0.05 was considered significant.Data were analyzed and results were presented as mean ± standard deviation. Student’s Figures C). Methylene blue did not reduce HT22 spontaneous apoptosis, compared with untreated control group . As predicted, serum deprivation for 24 h robustly increased both early and late apoptosis , which could be significantly inhibited by methylene blue in a dose-dependent manner .We firstly tested whether methylene blue exerted cytotoxicity in cells under normal culture condition. Mouse hippocampal HT22 cells treated with methylene blue ranging from 20 nM to 1 μM for 24 h did not increase either early or late apoptosis, suggesting that methylene blue has no significant pro-apoptotic effect within this range of concentration . Western blot results showed a dose-and-time-dependent increase of both LC3B-I and LC3B-II in methylene blue-treated cells, with the most robust increase being induced by 1 μM methylene blue . LC3B type II derives from processed LC3B type I and correlates well with autophagosome. Moreover, LC3B type II expression was increased in methylene blue-treated group, compared with serum deprivation group . Collectively, these data suggested methylene blue is an efficient macroautophagy inducer in HT22 cells.To determine whether methylene blue-induced macroautophagy, HT22 cells were treated with methylene blue at the dose range between 20 nM and 1 μM and macroautophagy was then detected with immunocytochemical staining and Western Blot. Serum deprivation was set as the positive control for macroautophagy. Anti-LC3B antibody staining displayed a relatively homogenous distribution of LC3B in HT22 cells, which was predominantly located in the cytoplasma, consistent with other reports . NotablyFigure 2D). To confirm that chloroquine inhibited methylene blue-induced macroautophagy, we tested both LC3B conversion and p62 level in treated cells. It is well accepted that in cells undergoing autophagic process, LC3B-II level is increased, while p62 level is reduced due to its role in targeting proteins into autolysosome. The mechanism underlying the inhibitory effects of chloroquine on macroautophagy is to impairing lysosomal acidification, thus protein degradation in autolysosome is blocked. Our data showed methylene blue induced an increased LC3B-II level as well as a decreased p62 level in HT22 cells in comparison with the untreated cells, indicating methylene blue indeed activates macroautophagy. In the presence of chloroquine, LC3B-II level was even higher than that in cells treated with methylene blue alone, along with a stabilized p62 level, suggesting chloroquine indeed inhibits protein degradation in autolysosomes .To test the role of macroautophagy in methylene blue-mediated neuroprotection, HT22 cells were simultaneously treated with methylene blue and serum deprivation for 24 h, and cell apoptosis were determined by flow cytometry. Consistently, methylene blue rescued HT22 cells from serum deprivation-induced apoptosis. However, the recuing effect was significantly blocked in the presence of a potent macroautophagy inhibitor-10 μM chloroquine . Chloroquine alone did not result in HT22 cell apoptosis. Collectively, these data suggested that macroautophagy play a critical role in methylene blue-mediated neuroprotection against serum-deprivation.Then, we tested whether methylene blue pre-treatment had protective effects. Compared with vehicle-treated group, methylene blue-pretreated HT22 cells were more resistant to apoptosis induced by serum deprivation, which could be blocked by chloroquine . Interestingly, the expression level of Bcl-2, a pro-survival and anti-apoptotic oncogenic protein, was robustly increased after methylene blue treatment in a dose-dependent manner .To determine the signal pathways involved in methylene blue-induced macroautophagy, HT22 cells were treated with methylene blue under normal culture condition and Western blots were performed using anti-Akt and anti-Erk antibodies. As shown in Figure 4A). To test whether AMPK pathway was critical for methylene blue-induced macroautophagy, cells were treated with 20 μM compound C, a selective AMPK inhibitor during the last 4 h of methylene blue treatment. Compound C co-treatment effectively inhibited methylene blue induced ACC phosphorylation, indicating that AMPK pathway was blocked . Moreover, LC3B type II level was significantly alleviated by compound C, suggesting that methylene blue-induced macroautophagy was indeed via AMPK pathway .5′ Adenosine monophosphate-activated protein kinase pathway has been characterized as another signal pathway that regulates macroautophagy , we detein vivo, we treated mice with methylene blue for 2 weeks, and AMPK activation and LC3B type II conversion in the hippocampus and cortex were then examined. As shown in Figures 5 and 6, no significant change of AMPKα protein level in the hippocampus and cortex was found upon methylene blue treatment. However, AMPKα phosphorylation was dramatically enhanced by methylene blue, especially in high dose-treated group. AMPKβ1 and AMPKβ2 were also significantly increased after methylene blue treatment in a dose-dependent manner. ACC phosphorylation was promoted in high dose group. Similar to in vitro data, phosphorylation of mTOR was not significantly altered in either methylene blue-treated group. Compared with vehicle-treated group, LC3B type II level was dramatically elevated, suggesting enhanced autophagosome formation in methylene blue-treated groups. In the SVZ, however, none of these signal pathways was altered after methylene blue treatment, indicating that the methylene blue might not affect neural stem/progenitor cells . LC3B conversion also showed differences in distinct brain areas. Methylene blue increased LC3B type II in hippocampi and cortex while slightly decreased LC3B type II in SVZ of methylene blue-treated mice. Taken together, these data demonstrated that methylene blue can induce macroautophagy in hippocampus and cortex through AMPK but not mTOR pathway in vivo.To determine whether methylene blue induces macroautophagy in vitro; (3) Methylene blue induces macroautophagy via enhancement of AMPK signal pathway in mouse hippocampus and cortex in vivo; (4) Methylene blue does not induce macroautophagy in neural stem/progenitor cells located at the mouse SVZ in vivo.In this study we find that (1) Methylene blue protects HT22 cells from serum deprivation-induced apoptosis through induction of macroautophagy; (2) Methylene blue induces macroautophagy via enhancement of AMPK pathway rather than inhibition of mTOR pathway Neurons in central nervous system are sensitive to inadequate serum supply. Studies have demonstrated the morphological and metabolic characteristics of neurons after serum deprivation . In the in vitro and in vivo. Consistently, Akt and Erk signaling, which has been proven to activate mTOR pathway, were not inhibited after methylene blue treatment. Bcl-2 was suggested to inhibit Beclin1-dependent macroautophagy. Methylene blue robustly increased Bcl-2 protein levels. Therefore, we speculated that Bcl-2 is not involved in methylene blue-induced macroautophagy. Rather, the anti-apoptotic Bcl-2 might be another mechanism contributes to the neuroprotectiove action of methylene blue.Previous studies have indicated that mTOR-related signal transduction is the key regulator of macroautophagy . Other sin vitro, whereas the changing pattern was more complicated in vivo, depending on the brain regions. In the hippocampus and cortex, the action of methylene blue on macroautophagy was similar to that in vitro, while no effect of methylene blue was observed in SVZ cells. Since SVZ consists of primitive neural stem/progenitor cells, we speculated that methylene blue-induced AMPK activation mainly occurs in mature neurons. Elaborating studies have shown that methylene blue act as an alternative electron carrier in the mitochondrial electron transport chain to enhance ATP production while energy is in short. So it is unlikely that AMPK activation in methylene blue-treated cells was due to increased AMP/ATP ratio. Promoted ACC phosphorylation indicated AMPK pathway was indeed activated. Methylene blue-induced AMPK activation was accompanied by higher levels of LC3B type II, the activated form of LC3 participating autophagosome formation. Furthermore, when AMPK inhibitor was administrated with methylene blue, ACC phosphorylation was reverted to basal level together with reduced LC3B type II level.5′ Adenosine monophosphate-activated protein kinase plays as a master regulator of cellular energy homeostasis . The kinTaken together, the present study suggests that methylene blue exert protective effect against serum deprivation, at least partly, through enhancing macroautophagy. In addition, our study demonstrated that activation of AMPK pathway, but not mTOR, is involved in the methylene blue-induced macroautophagy.The University of North Texas Health Science Center has filed a PCT patent application entitled “Compounds that enable alternative mitochondrial electron transfer.”


The results of the genital human papillomavirus (HPV) detection in 439 cervical samples by cervical cytology were compared with sequencing analysis and a newly developed HPV genotyping 9G membrane test. The excellent sensitivity and specificity of the HPV genotyping 9G membrane test was assured by a signal to noise ratio of more than 300 and a target hybridization to non-target hybridization ratio of 300 ~ 400 at 25 °C. The final results can be obtained in 29 min by simple loading of the hybridization and washing solutions and scanning the membranes without any drying steps or special handling. The 100% identical results of the HPV genotyping 9G membrane test with sequencing results in 439 clinical samples demonstrate significant clinical application for this test. HPV genotyping 9G membrane tests can identify and discriminate five HR-HPV genotypes which are prevalent in almost 87% of cervical cancer cases. Its simple handling makes the HPV genotyping 9G membrane test a very convenient platform for accurate HPV genotyping. Genital human papillomavirus (HPV) is the most common sexually transmitted infection in the United States, with an estimated 6.2 million people becoming newly infected every year . CervicaHowever, traditional methods for HPV detection, such as morphological and immunological methods, lack the ability to detect specific HPV types . Real-tiThe high mortality rate in HPV infected patients demands the urgency to develop a rapid and field-deployable HPV genotyping test . IdeallyRecently, we have reported the 9G DNAChip technology  for the The HPV genotyping 9G membranes for the rapid detection and discrimination of the HPV genotypes were tested in 439 clinical samples. It is for the first time that the genotyping results are obtained by the direct hybridization of the Cy5 labeled PCR products to the immobilized probes on the membrane. The HPV genotyping 9G membranes show high sensitivity, 100% target-specific hybridization, discrimination of the genotypes with a ratio of 360:1, at 25 °C in 28 min, and does not need a complex and expensive instrumentation. The HPV genotyping 9G membrane test can effectively detect the five HR-HPV genotypes which are prevalent in cervical cancer cases. The HPV genotyping 9G membrane is basically designed for the detection and discrimination of the five HR-HPV genotypes. Moreover, the HPV2 genotyping 9G membrane can screen the presence or absence of the other 14 HR-HPV genotypes.The primed PCR product was added to the sequencing reaction mixture. Sequencing was performed bidirectionally with the BigDye3 terminator cycle sequencing kit (PE Applied Biosystems) using ABI PRISM 310 Genomic Analyser (PE Applied Biosystems) at a dispensing pressure of 600 mbar with 8-ms open times and 65-s cycle times. The sequencing procedure was carried out by stepwise elongation of the primer strand upon cyclic dispensation of the different deoxynucleoside triphosphates (Amersham Pharmacia Biotech). A CCD camera detected the light output resulting from nucleotide incorporation. The data were obtained in a graphic format . Out of The HPV genotyping 9G membranes consist of the five HR-HPV type specific probes . Whereas, the HPV2 genotyping 9G membrane consist of the 14 HR-HPV type specific probes . The 5 µl of PCR product was subjected to agarose gel electrophoresis, using a 2% agarose gel, the product size of HPV DNA was found to be 250 base pairs (bp). etc.) was loaded into the sample loading port on the HPV genotyping 9G membrane strip. If necessary another 110 μL of hybridization mixture containing Cy5 labeled PCR product was loaded on the HPV2 genotyping 9G membrane strips. The hybridization, washing and scanning procedure was followed as mentioned earlier. Each experiment was done more than three times. HPV amplicons can be hybridized with type-specific oligonucleotide probes on HPV genotyping 9G membrane and can visualized on the graph after analysis of results  and (ii) results, the use of the HPV2 genotyping 9G membrane test is not necessary as the HPV genotyping 9G membrane test can either detect the absence of HPV genotype or presence of at least one HR-HPV genotype out of five HR-HPV genotypes. However, in the case of result (iii), the hybridization mixture containing same PCR product should be loaded on the HPV2 genotyping 9G membrane test for further detection of the HR-HPV genotype out of 14 HR-HPV genotypes.In the case of the HPV2 genotyping 9G membrane test, two possible results as shown in the Interestingly, the excellent specificity of the probes immobilized on the HPV genotyping 9G membranes ensured a SBR of more than 300  which isThe results of the HPV genotyping by the HPV genotyping 9G membrane tests were compared with the results of the HPV genotyping by the sequencing analysis and the cervical cytology in the 439 clinical samples, in which specific HPV genotypes were, detected . Out of Out of 140 HPV positive cases, 83% were detected to be HR-HPV genotypes and the 17% were detected to be NOT HR-HPV genotypes. The distribution of the HPV genotypes in the 439 clinical samples is demonstrated in The comprehensive analysis of the results presented in The accuracy of the HPV genotyping 9G membrane tests for the detection of the HPV genotypes in 439 clinical samples was calculated from the sensitivity (true positive rate) and specificity (true negative rate) of 95% confidence intervals (CI). Considering the fact that HPV genotyping 9G membrane tests showed 100% identical results with the sequencing analysis, the samples that were positive by sequencing analysis and the HPV genotyping 9G membrane tests were defined as true positive and specimens negative by both of these methods were defined as true negative. The sensitivity and specificity were calculated by assuming that the sequencing analysis of the clinical samples was correct.Interestingly, the excellent specificity of the probes immobilized on the HPV genotyping 9G membranes ensured the SBR of more than 300  which isThe 100% clinical sensitivity as well as 100% clinical specificity was observed for the HPV genotyping by the HPV genotyping 9G membrane tests. The clinical sensitivity and specificity of the cervical cytology was 67% and 80%, respectively. It is evident from the results of HPV genotyping 9G membrane test that many clinical samples designated as normal in cervical cytology were found to be high risk type HPV genotypes (20%). The low sensitivity and specificity of cervical cytology may lead to improper diagnosis. On the contrary, the HPV genotyping 9G membrane test shows profound accuracy in genotyping HPVs in clinical samples.The major disadvantage of commercial DNA chip based HPV genotyping tests is that they require a complicated washing and drying step which requires highly trained professionals. However, in the case of HPV genotyping 9G membrane tests, hybridization solution and washing solution are directly loaded on the membranes. The final results can be obtained by scanning the membranes without any drying steps or special handling. These simple handling steps make HPV genotyping 9G membrane tests a very convenient platform for accurate HPV genotyping.The 100% sensitivity and specificity in the detection and discrimination of the HR-HPV genotypes establish the accuracy of HPV genotyping 9G membrane tests in the clinical diagnosis of HPV infections. The following factors probably contribute to proficient genotyping by the presented test: (i) hybridization, washing, and direct scanning at 25 °C, (ii) high SBR of 300 as compared to those of 2.5–5 for the other tests, (iii) the 100% target-specificity in terms of the 100% identical results with sequencing analysis.The results for sequencing of the HPV genotypes HPV-16 (26%), HPV-52 (17%), HPV-58 (14%), HPV-39 (8%), HPV-56 (7%), HPV-18 (5%), and HPV-31 (5%) were 100% identical with the HPV genotyping 9G membrane test. The 67%  of HR-HPV genotypes which were found to cause HSIL were detected by the HPV genotyping 9G membrane test and the remaining 13% (HPV58) was detected by the HPV2 genotyping 9G membrane test. Similarly, the 60%  of HR-HPV genotypes which were found to cause LSIL were detected by the HPV genotyping 9G membrane test and the remaining 40%  were detected by the HPV2 genotyping 9G membrane test. 1 copies and other HPV genotypes including HPV45 is 102 copies [It is interesting to note that the results of cervical cytology demonstrates that, out of 439 samples, 362 were normal and others were ASC-US (47 samples), ASC-H (9 samples), LSIL (9 samples), and HSIL (12 samples) . However2 copies . HPV genotyping tests based on the HPV genotyping 9G membrane tests are accurate methods for detection and genotyping of HPV. The genotypes of HPV genotyping 9G membrane tests were in 100% agreement with genotypes of HPV DNA sequencing. The HPV genotyping 9G membrane tests have proved that the preliminary findings of the cervical cytology of HPV infected samples may lead to the false detection of HPV. Out of 362 clinical samples, which were designated as normal (HPV negative) by cervical cytology, the 72 samples (20.4%) were identified as HPV positive in the HPV genotyping 9G membrane tests.The TaqMan PCR or other PCR techniques also represent a sensitive method for the detection of HPV DNA. Currently, standard nested HPV PCR can be performed using degenerative primers followed by direct sequencing of the PCR product. Alternatively, PCR products can be hybridized to DNA of known HPV types, to determine the type of HPV present in the PCR amplified sample . Both assays are time consuming and often associated with false positive results.The present HPV genotyping 9G membrane test is designed for the detection and discrimination of the five HR-HPV genotypes. However, it can effectively identify and discriminate five HR-HPV  genotypes which are prevalent in almost 87% of cervical cancer cases. Moreover, the HPV2 genotyping 9G membrane can screen the presence or absence of the other 14 HR-HPV genotypes in clinical samples.However, the HPV genotyping 9G membrane tests take only 29 min for highly specific detection of HPV genotypes. Moreover, the advantage of HPV genotyping 9G membrane test is that it uses a simple hybridization and washing step which allows it to be used in small laboratories, and does not need highly trained professionals.. All oligonucleotides were purchased from Bioneer, Korea. All chemicals were purchased from Sigma-Aldrich Chemicals, Korea. All washing solvents for the substrates are of HPLC grade from SK Chemicals, Korea. Ultrapure water (18 M Ω/cm) was obtained from a Milli-Q purification system .Glass fiber membrane (2.5 × 7.5 cm) was purchased from Whatman, Springfield, UKTM .Oligonucleotides were lined using dispenser . Hybridization was done at 25 °C and no special instrument is required. The fluorescence signal intensities were measured and analyzed by BMT Membrane Reader4Cl, (b) Blocking buffer solution (pH = 7.4): 0.5% milk casein in 4× SSC, (c) Hybridization buffers (pH = 7.4): 25% Formamide, 0.1% Triton X-100, 6x SSC, d) Washing buffer solution (pH = 7.4): 0.1% SDS in 4× SSC.(a) Immobilization solution (pH = 7.4): 15% glycerol, 50 mM butyl amine, 600 mM NHThe HPV genotyping 9G membranes were obtained by slight modification in the 9G DNAChip technology used for the fabrication of the 9G DNAChips. Unlike the nitrocellulose membranes  in otherAfter immobilization, the membranes were soaked in the blocking solution and then dried to generate the HPV genotyping 9G membrane . SimilarTM to obtain final results. Each experiment was done more than three times. The genotyping results can be obtained in 29 min by using HPV genotyping 9G membrane tests.The hybridization buffer was prepared by mixing the 20 mL of hybridization solution and 600 μL of Cy5-HC-T1 (60 fmol/μL). A final 240 μL of hybridization mixture was prepared by mixing the 220 μL of the hybridization buffer and 20 μL of Cy5 labeled PCR product of HPV genotype . Out of 240 μL, the 110 μL of this hybridization mixture was loaded on sample loading port on the HPV genotyping 9G membrane strip and allowed to hybridize for 20 min at 25 °C. After hybridization, washing solution was loaded into the washing port and allowed to stand for 8 min. Then, the 9G membranes were scanned by BMT Membrane ReaderThe samples were collected consecutively from 439 Korean women who visited the Department of Obstetrics and Gynecology, CHA Clinic, South Korea. The samples were collected by scraping the uterine cervical canal with a small cytobrush after Pap smear, and the brush was put into a 15 mL centrifuge tube containing phosphate-buffered saline. The specimens were collected as part of an informed consent protocol approved by the clinical studies committee of the CHA Bundang hospital, South Korea.The cervical cytology was performed on all 439 cervical samples. Out of 439 samples, the HPV positive results were found in 77 samples and the remaining 362 samples were found as normal (No HPV detected) . The HPV+/GP6+ primer set-mediated PCR (GP+-PCR) are the most frequently used amplification systems for the detection of HPV DNA in clinical samples, amplifying DNA fragments in the conserved L1 region with approximately 450 bp and 150 bp, respectively. Further, type-specific PCR primer sets allow the identification of individual genotypes. The MY11/ GP6+ primer set consists of a fixed nucleotide sequence for the forward and reverse primers, respectively, and detects a wide range of HPV types by using a lowered annealing temperature during PCR [The MY09/MY11 primer set-mediated PCR (MY-PCR) and the GP5ring PCR ,20.Taq DNA polymerase in an amplification buffer containing 2mM MgCl2, and a tracking dye (Cy5). All tubes were incubated for 2 min at 50 °C before PCR was started. Amplification was performed with the following steps: pre-denaturation for 5 min at 94 °C, 45 cycles of 30 s each for the denaturation at 94 °C; 45 cycles of 30 s each for annealing at 65 °C, 45 cycles of 30 s each for elongation at 72 °C, and an final elongation step of 7 min at 72 °C. 5 µL of PCR product was subjected to agarose gel electrophoresis, using a 2% agarose standard run in 1X Tris borate EDTA. 10 μl of this Cy5 labeled PCR product was used for the further hybridization experiments in the HPV genotyping 9G membrane tests for the HPV detection and genotyping. The whole procedure was followed for each of the 439 clinical samples.The whole HPV genomic DNA extracted from clinical samples was amplified by duplex PCR to generate amplicons. HPV DNA was amplified with primers RP and FP . The PCRThe excellent sensitivity and the specificity of the HPV genotyping 9G membrane tests were assured by a signal to noise ratio of more than 300 and a target hybridization to non-target hybridization ratio of 300 ~ 400. In the case of HPV genotyping 9G membrane tests, the hybridization and washing solutions are directly loaded on the membranes. The final results can be obtained by scanning the membranes without any drying steps or special handling. These simple handling steps make the HPV genotyping 9G membrane tests a very convenient platform for accurate HPV genotyping.Furthermore, in this study, the accuracy of HPV genotyping 9G membrane tests for HPV genotyping could be certified by comparison with the sequencing data, which is found to be 100% identical in all cases. The HPV genotyping 9G membrane tests demonstrate a clinical value in decision-making as they can identify and discriminate HR-HPV genotypes which are prevalent in almost 87% of cervical cancer cases. The efficient detection and discrimination of the 439 clinical samples make HPV genotyping 9G membrane tests a promising diagnostic tool for accurate HPV genotyping.


We conducted a systematic review and meta-analysis to assess efficacy and safety of prophylactic HPV vaccines against cervical cancer precursor events in women.2 statistic.Randomized-controlled trials of HPV vaccines were identified from MEDLINE, Cochrane Central Register of Controlled Trials, conference abstracts and references of identified studies, and assessed by two independent reviewers. Efficacy data were synthesized using fixed-effect models, and evaluated for heterogeneity using ISeven unique trials enrolling 44,142 females were included. The fixed-effect Relative Risk (RR) and 95% confidence intervals were 0.04 (0.01-0.11) and 0.10 (0.03-0.38) for HPV-16 and HPV 18-related CIN2+ in the per-protocol populations (PPP). The corresponding RR was 0.47 (0.36-0.61) and 0.16 (0.08-0.34) in the intention-to-treat populations (ITT). Efficacy against CIN1+ was similar in scale in favor of vaccine. Overall vaccines were highly efficacious against 6-month persistent infection with HPV 16 and 18, both in the PPP cohort , and the ITT cohorts . There was limited prophylactic effect against CIN2+ and 6-month persistent infections associated with non-vaccine oncogenic HPV types. The risk of serious adverse events  or vaccine-related serious adverse events  did not differ significantly between vaccine and control groups. Data on abnormal pregnancy outcomes were underreported.Prophylactic HPV vaccines are safe, well tolerated, and highly efficacious in preventing persistent infections and cervical diseases associated with vaccine-HPV types among young females. However, long-term efficacy and safety needs to be addressed in future trials. Oncogenic HPV infection is the necessary cause of cervical cancer . WorldwiTwo meta-analyses have been published evaluating prophylactic L1 VLP-based HPV vaccine efficacy and safety ,4. Few rst, 2009, using a combination of index terms: "Human Papillomavirus", "Papillomavirus vaccines", "Randomized controlled trials", and "Controlled clinical trials". Additional relevant studies were sought through hand search of conference abstract books from IPVS  from 2006 to 2009, and bibliographies of obtained studies.A systematic search of MEDLINE, Cochrane Library and the Cochrane Central Register of Controlled Trials was conducted to identify reports of RCTs of prophylactic HPV vaccines published up to July 31RCTs published in English of L1 VLP-based HPV vaccines that were conducted among women and presented measurement of prophylactic efficacy against HPV infection or diseases of interest were included. Trials reporting male vaccination or therapeutic vaccination were excluded. Additionally, we excluded ad hoc subgroup analyses of existing RCTs or combined analyses of multiple RCTs. In the case a RCT reported both interim and final analyses, data from final analyses with complete follow-up were used.High-grade cervical lesions or worse (CIN2+), including Cervical Intraepithelial Neoplasia (CIN) grade 2-3, Adenocarcinoma in Situ (AIS), and cervical carcinoma, was the recommended endpoint for establishing efficacy of prophylactic HPV vaccines by WHO and consistently reported in most trials. It was, therefore, chosen as the primary endpoint for efficacy in this review. As persistent HPV infection is an obligate precursor of CIN2-3 and cervical cancer , type-spData were extracted by two independent reviewers (BL and AK) using a standardized data extraction form. Any discrepancies were resolved by consensus or in consultation with a third reviewer. We extracted detailed information on trial design, inclusion/exclusion criteria, participant characteristics, vaccine and placebo administered, trial endpoints, efficacy populations, and methodological quality from all included trials.2 statistics  and expressed as percentage. Three types of populations were defined for efficacy analyses: The intention-to-treat population (ITT), the modified intention-to-treat population (MITT), and the per-protocol population (PPP). Vaccine efficacy was assessed in ITT and PPP cohorts respectively. Efficacy estimates from MITT cohorts were used in the absence of ITT estimates. Vaccine safety was primarily evaluated in ITT cohorts among women who received at least one injection of vaccine or control and had available follow-up, except in one trial ,9. A fixatistics . Sensitit method ,12. The t method . All anat method  followint method .Study identification and selection was demonstrated in the flow diagram in Figure Characteristics of RCTs included in the current review are summarized in Table The mean age of participants for the majority of trials was approximately 20 years except in the trial of Munoz and associates where older females were recruited (mean age 34) , indicated no significant publication bias.The Begg and Egger funnel plot .The pooled RR for 4 RCTs enrolling 28,639 participants was 0.47 (95% CI: 0.36-0.61) in ITT cohorts, corresponding to a pooled efficacy of 53% and indicating a statistically significant benefit with vaccine use. However, there was significant heterogeneity among pooled studies  was 0.04 (95% CI: 0.01-0.11), showing a statistically significant reduction of 96% in CIN2+ incidence due to vaccination.Analysis using ITT cohorts showed a statistically significant benefit of vaccination with a pooled RR  of 0.16 (95% CI: 0.08-0.34), translating to an 84% protection against HPV 18 associated CIN2+ for vaccine recipients.Two of the three trials included in the ITT analyses provided extractable data for the PPP analyses. The overall RR for PPP cohorts  was 0.10 (95% CI: 0.03-0.38), equivalent to a 90% protection against HPV 18 associated CIN2+ for vaccine recipients compared with control recipients.2 = 76%). The summary RR for PPP cohorts  was 0.02 (95% CI: 0.00-0.11), indicating a 95% reduction in CIN1+ incidence due to vaccine use.Vaccines of different compositions demonstrated a statistically significant level of protection against HPV 16-associated CIN 1+ in ITT cohorts. The pooled RR  was 0.43 (95% CI: 0.33-0.58) corresponding to a 57% reduction in CIN1+ incidence for vaccine recipients compared with control recipients. However, there was statistically significant heterogeneity among included trials  of 0.22 (95% CI: 0.10-0.44), translating to an efficacy of 78%. Only one of three trials provided efficacy data for PPP cohort, reporting a RR  of 0.03 (95% CI: 0.00-0.51), a 97% reduction.Persistent infection of ≥6 months was defined as the detection of same HPV DNA at two or more consecutive visits over a minimum of 4 months from the beginning of case counting. While three RCTs reported efficacy information for PPP cohorts, only two provided information for ITT cohorts. The pooled RR  for the risk of 6-month persistent HPV 16 infection in ITT cohorts was 0.15 (95% CI: 0.10-0.23), equivalent to an 85% of risk reduction for vaccine recipients compared with control recipients.A statistically significant protection derived from vaccine use was observed in PPP cohorts. The pooled RR  was 0.06 (95% CI: 0.04-0.09), corresponding to a 94% lower risk of persistent HPV 16 infection for vaccine recipients in PPP cohorts.Vaccines conferred a statistically significant protection against 6-month persistent HPV 18 infection, with a pooled RR of 0.24 (95% CI: 0.14-0.42) for ITT cohorts  and a RR of 0.05 (95% CI: 0.03-0.09) for PPP cohorts , equivalent to a 76% and 95% protection derived from vaccination, respectively.2 = 74%). The overall RR was 0.58 (95% CI: 0.43-0.77) for PPP cohorts , indicating a ~40% cross-protection against non-vaccine oncogenic HPV-associated CIN2+.In ITT analyses, vaccines demonstrated statistically significant reduction (p = 0.003) in the risk of CIN2+ associated with non-vaccine oncogenic HPV types with a pooled RR  of 0.79 (95% CI: 0.67-0.92) Figure . HoweverTwo RCTs demonstrated comparable level of protection against 6-month persistent HPV 31/33/45/52/58 infection in ITT and PPP cohorts, respectively Figure . The poo2 = 94%; PPP: Cochrane's Q, p < 0.0003, I2 = 93%).When cross-protection against 6-month persistent infection was examined by HPV type Figure , prophylAdverse events (AEs) were monitored by the use of daily vaccination report cards within 15 or 30 days of injection, as well as solicitation throughout the study. Occurrence of AEs was reported in all RCTs. Pain at injection site was the most frequently reported AE ranging from 83.0 - 93.4% in vaccine groups and 75.4 - 87.2% in control groups. Headache and fatigue were the most common vaccine-related systemic AEs observed in approximately 50-60% of all participants. Serious AE reported included abnormal pregnancy outcomes, blood and lymphatic system disorder, hepatobiliary disorder, immune system disorder, cardiac and vascular disorder, gastrointestinal disorder, musculoskeletal and connective tissue disorder, nervous system disorder, psychiatric disorder, renal and urinary disorder, reproductive system and breast disorder, respiratory, thoracic and mediastinal disorder, skin and subcutaneous tissue disorder, neoplasm, infection and infestation, injury, poisoning and procedural complications. The most commonly reported serious AEs were abnormal pregnancy outcomes such as abnormal infant and spontaneous abortion. The pooled RR for participants experiencing one or more serious AEs was 1.00 (95% CI: 0.91-1.09) suggesting a statistically insignificant difference in the risk of serious AEs between vaccine and control groups Figure .Serious AEs that were judged to be related to injection included bronchospasm, gastroenteritis, headache, hypertension, injection-site pain, decrease in joint movement at injection site, hypersensitivity to injection, chills, headache and fever. Four of the seven trials reported zero injection-related serious AEs Figure . Among t2 = 59%, p = 0.09; CIN1+; I2 = 0%, p = 0.70). The previously observed heterogeneity and lower efficacy reported by the FUTURE trials was likely due to inclusion of a larger proportion of trial participants already infected with vaccine HPV types at baseline in the ITT cohorts, some of whom may have progressed to cervical neoplasia during the follow-up period.Sensitivity analysis was performed to identify potential sources of heterogeneity that was observed in the ITT analyses of CIN1+ and CIN2+ associated HPV 16. The included trials did not differ by methodological qualities in terms of allocation concealment, blinding, effect size, power calculation, and participant withdraw and drop-out. Therefore, heterogeneity was unlikely attributed to the methodological quality. We further examined the heterogeneity among included trials according to study characteristics including study population, inclusion/exclusion criteria, intervention, comparator, endpoints chosen and efficacy populations defined, as well as participant baseline characteristics such as age, HPV prevalence and lifetime number of sex partners. The pooled efficacy estimates for FUTURE trials were significantly lower than those for all other RCTs . With the FUTURE trials excluded, the observed heterogeneity dissipated for either endpoint  to vaccine HPV types at enrollment and remained DNA-negative through the regimen with no protocol violation. In addition, case counting for ITT or MITT analyses usually started on day 1, instead of one month post dose three in most PPP analyses. The excessive cases observed in ITT cohorts compared with those in PPP cohorts were likely the results of incomplete vaccine regimen and the inclusion of prevalent cases of HPV infection and low-grade cervical diseases at enrollment.The ITT cohort in the vaccine trials mimics young women in the general population who may have been exposed to vaccine type HPV infection and have less than perfect compliance with vaccination protocol, whereas PPP cohorts approximates pre-sexually active young adolescents naïve to vaccine type HPV with perfect or nearly perfect compliance. The young adolescent group becomes susceptible soon after their initiation of sexual activities, even if they are not engaged in penetrative sexual intercourse ,20. The While sexually naïve young women may derive greater public health benefit from HPV vaccination, vaccine appears to be of benefit to a broad age range of women many of whom may have acquired transient infections in the past or had active infection at the time of vaccination. Preliminary data from the quadrivalent vaccine trial of Muñoz and associates  enrollinWhether prophylactic vaccines offer long-term protection remains an important unanswered question. Two included trials offered an extended follow-up of 53 months and 60 months ,23 and bOur results demonstrated statistically significant but limited protection against CIN2+ associated with non-vaccine-type oncogenic HPV that are phylogenetically related to HPV 16 and 18 in both ITT and PPP cohorts (RR: 0.79 in ITT and 0.58 in PPP). Limited cross-protection against persistent infection of ≥6 months was consistently observed for HPV 31, and to a lesser extent against persistent HPV 45 and HPV 33 infection. No statistically significant protection was detected against persistent infection with HPV 52 and 58. The added benefit of cross-protection may result in further reductions in incidence of cervical cancer and precancerous lesions following vaccination. However, whether cross-protection remains present in long-term follow-up, and if present, how the efficacy trend for non-vaccine types compares with that of vaccine HPV types has yet to be determined.Results of the safety assessment indicated that injection-related local and systemic symptoms were generally mild. The most commonly reported serious AEs were abnormal pregnancy outcomes. Serious AEs that were considered to be vaccine-related were rare. It was noted that data on abnormal pregnancy outcomes were underreported, only available in three of the seven trials included ,17,25. TThere are several limitations to the evidence presented here. We were not able to evaluate prophylactic efficacy against anogenital warts, vulvar or vaginal diseases associated with vaccine HPV due to the common use of composite endpoints in individual RCTs that combined infections, lesions and cancers of various anogenital sites, and were often incomparable across RCTs ,18,23. IIn conclusion, our review demonstrated that VLP-based prophylactic HPV vaccines are highly efficacious in preventing persistent infection and cervical diseases associated with vaccine HPV types among young female adults. The vaccines were safe and generally well tolerated. Vaccination of adolescent girls prior to sexual debut appeared to be the most effective public health measure for prevention of cervical diseases and cancer. Questions related to long-term efficacy and safety have yet to be addressed.Authors declare that (1) No author have received support from any company for the submitted work; (2) XC serves as a consultant to Sanofi Pasteur MSD Ltd. and GlaxoSmithKline, and ARG serves an advisory board member to Merck & Co. Inc.; (3) their spouses, partners, or children have no financial interests that may be relevant to the submitted work; and (4) all authors have no non-financial interests that may be relevant to the submitted work.BL was responsible for conception and design of the study, conduct of analysis, interpretation of data, and drafting and revision of the manuscript;         AK was involved in design of the study, conduct of analysis, interpretation of data and critical revision of the manuscript; XC and ARG participated in design of the study, interpretation of data, and critical revision of the manuscript. All authors have read and approved the final manuscript. Requirement on Ethics Approval: As this analysis is a secondary analysis of published data and uses no identifiable patient information, ethical approval is not required. Data Access: All authors of this manuscript had full access to all of the data  in the study and can take responsibility for the integrity of the data and the accuracy of the data analysis.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2334/11/13/prepubFigure 1. Assessment of Publication Bias for the Primary Endpoint, CIN2+ Associated with HPV 16 and 18. Funnel plot for assessment of publication bias.Click here for fileFigure 2. Sensitivity Analysis for Select Endpoints with Significant Heterogeneity. Forest plots showing results of subgroup analysis for select endpoints.Click here for file


Objective. To compare knowledge and attitudes of human papillomavirus (HPV) and the vaccine between different cultures of African descent. Methods. A cross-sectional survey of 555 African-Americans and Afro-Caribbeans residing in the US and the Bahamas (BHM) was conducted. Results. General knowledge about HPV and the HPV vaccine differed between the two countries significantly. Bahamian respondents were less likely to have higher numbers of correct knowledge answers when compared to Americans . Older age, regardless of location, was also associated with answering fewer questions correctly . Attitudes related to HPV vaccination were similar between the US and BHM, but nearly 80% of BHM respondents felt that children should not be able to receive the vaccine without parental consent compared to 57% of American respondents. Conclusions. Grave lack of knowledge, safety and cost concerns, and influence of parental restrictions may negatively impact vaccine uptake among African-American and Afro-Caribbean persons. Interventions to increase the vaccine uptake in the Caribbean must include medical provider and parental involvement. Effective strategies for education and increasing vaccine uptake in BHM are crucial for decreasing cervical cancer burden in the Caribbean. Cervical cancer (CC) is listed among the top five cancers that affect women globally. According to the International Agency for Research on Cancer (IARC), in 2008 there were 530,000 new cases of cervical cancer worldwide, with 85% of the disease burden occurring in developing countries. Caribbean women are at an increased risk of dying from CC, which is the second most frequent cancer among women and also ranks as the third most frequently diagnosed cancer in both sexes . In the Gardasil (against HR HPV types 16 and 18 as well as low-risk types 6 and 11) for all females aged 9 through 26. In 2009, the FDA also approved the use of this vaccine in males aged 9 through 26 [Cervarix, which aims to protect against HR HPV types 16 and 18 only, was approved by the FDA in the Fall of 2009 for females aged 10 through 25 [Persistent high-risk (HR) human papillomavirus (HPV) infection, specifically HPV types 16 and 18, has been linked to the development of CC, anogenital cancers, and oropharyngeal cancers , 5. In 2rough 25 . The CenP > 0.1) [ Reasons as to why disparity exists for HPV vaccine uptake and completion remain unclear in the literature. Some studies attribute racial disparity to income level differences, while others believe it is due to lack of knowledge –12. The P > 0.1) . Cultural beliefs or perceptions and lifestyle are distinct between persons born in the US and those born in other countries –16. WhilThe original study population consisted of 202 volunteer participants from the University of Pittsburgh and Hampton University . The recThe questionnaire used in this study consisted of demographic questions pertaining to age, race, sex, place of birth, religion, household income, level of education, health insurance status, parental status, and number of children. The study instrument included measures of HPV knowledge and the safety, efficacy, and impact of the HPV vaccine. A content assessment of the survey was conducted prior to the launch of the study .t-tests. Adjusted prevalence of correct answers to HPV and HPV vaccine knowledge questions was calculated and stratified by geographic location after adjusting for age, level of education, marital status, parental status, health insurance status, and income level. Ordered logistic regression was performed to assess differences in response to knowledge questions pertaining to HPV and HPV vaccination between the two groups, adjusting for demographic variables and the covariates previously mentioned. Statistical analysis was performed using STATA version 10.1 software . A P value of ≤0.05 was considered statistically significant.Descriptive statistics were generated for demographic and knowledge variables according to the place of residence for study participants. Fisher's exact test was used to determine statistical significant differences between proportions; differences in continuous variables were assessed using two-sample n = 23) were immigrants and not born in their current country of residence. Twenty-five percent of the participants were male, and 75% were female; there was no difference in gender between the two geographic locations. In both locations, the majority of the participants were between the ages of 18 and 35, with a higher proportion of this age group in the US sample compared to the BHM . Eighty-four percent of the US population was single compared to only 42% of the BHM population. Parenthood also demonstrated differences, where more Bahamians tended to be parents . The correlation between parental status and marital status also differed between the US and Bahamas. The majority of Bahamian parents were married (45%), while the majority of US parents were single (41%). All US respondents completed high school, while 11.5% of Bahamians did not. US participants were more likely to have attended or completed college . Income levels varied modestly between groups up to the US $50,000 mark; 40.7% of US respondents fell into this bracket compared to 11.7% of Bahamian respondents reporting this as their income.A total of 555 Black participants residing in the US and the Bahamas (BHM) completed the survey (41% from the US and 59% from the BHM ). BetweeP < 0.001). This difference in knowledge remained consistent for 6 out of the 10 questions related to HPV knowledge, while there was no difference in knowledge for four specific questions. Although the majority of respondents from both locations knew that only women could develop cervical cancer, lower proportions of respondents knew that HPV causes genital warts (28.2% of US and 33.2% of Bahamians) and that genital warts are not caused by the same HPV types that cause cervical cancer (13.8% of Americans and 7.5% of Bahamians).General knowledge about HPV and the HPV vaccine differed between the two countries significantly . When asP = 0.021).For the questions related to HPV vaccination , the majP = 0.013). More than half of both populations knew that, in spite of vaccination, annual Pap smears were still needed to screen for cervical cancer according to the recommendation at that time. Even so, a significantly lower proportion of Bahamians answered this question correctly . There was no difference between the two groups when asked what age group was eligible to receive the vaccine; less than half of each group answered with the correct age group, 9–26 .For two of the three questions assessing knowledge about the HPV vaccine , Bahamia For the participants that had previous knowledge of the HPV vaccine, the source of this information was similar between the two groups . For theWhen asked whether or not the HPV vaccine should be given to both boys and girls, both US and BHM participants had mixed views; however, the majority of both groups agreed that both genders should receive the vaccine . For botA number of international studies have focused on knowledge and attitudes related to HPV and the HPV vaccine. However, a majority of these studies have been conducted in Europe, Asia, and Africa –23, with More than half of participants from each location were aware that a vaccine for HPV is available; however Bahamian participants were less aware of this. Studies that focused on populations of African descent, outside of the US, report a similar lack of knowledge in regard to vaccine availability , 26, and Published literature suggests that in most cases, after parents were well informed about the risks and benefits of the vaccine, they were willing to vaccinate their children , 19, 28,P < 0.001). This difference may suggest that Americans have a higher self-worth in regard to their health, which is not the case when it comes to children. Hughes reports that children considered themselves to be passive participants when determining their course of medical care [ In general, attitudes related to HPV vaccination were similar between the US and BHM but differed when the two populations were asked if a well-informed child should be able to request vaccination at sexual health clinics without parental consent. US participants were more likely to agree with this statement . Respondents who agreed with the statement about children being able to decide about their own vaccination tended to be younger people (<35 years), regardless of geographic location (Lack of knowledge in regard to HPV and the HPV vaccine is not a problem that is limited to peoples of African descent but extends to ethnicities within developing countries all over the world. Regardless of geographic location, Whites have consistently displayed higher levels of knowledge concerning HPV and the vaccine when compared to other ethnic groups , 26. Eff


The distribution of HPV genotypes, their association with rigorously confirmed cervical precancer endpoints, and factors associated with HPV infection have not been previously documented among HIV-infected women in India. We conducted an observational study to expand this evidence base in this population at high risk of cervical cancer.HIV-infected women (N = 278) in Pune, India underwent HPV genotyping by Linear Array assay. Cervical intraepithelial neoplasia (CIN) disease ascertainment was maximized by detailed assessment using cytology, colposcopy, and histopathology and a composite endpoint.CIN2+ was detected in 11.2% while CIN3 was present in 4.7% participants. HPV genotypes were present in 52.5% (146/278) and ‘carcinogenic’ HPV genotypes were present in 35.3% (98/278) HIV-infected women. ‘Possibly carcinogenic’ and ‘non/unknown carcinogenic’ HPV genotypes were present in 14.7% and 29.5% participants respectively. Multiple (≥2) HPV genotypes were present in half (50.7%) of women with HPV, while multiple ‘carcinogenic’ HPV genotypes were present in just over a quarter (27.8%) of women with ‘carcinogenic’ HPV. HPV16 was the commonest genotype, present in 12% overall, as well as in 47% and 50% in CIN2+ and CIN3 lesions with a single carcinogenic HPV infection, respectively. The carcinogenic HPV genotypes in declining order of prevalence overall included HPV 16, 56, 18, 39, 35, 51, 31, 59, 33, 58, 68, 45 and 52. Factors independently associated with ‘carcinogenic’ HPV type detection were reporting ≥2 lifetime sexual partners and having lower CD4+ count. HPV16 detection was associated with lower CD4+ cell counts and currently receiving combination antiretroviral therapy.HPV16 was the most common HPV genotype, although a wide diversity and high multiplicity of HPV genotypes was observed. Type-specific attribution of carcinogenic HPV genotypes in CIN3 lesions in HIV-infected women, and etiologic significance of concurrently present non/unknown carcinogenic HPV genotypes await larger studies. Human immunodeficiency virus (HIV)-infected women in India and other developing countries are living longer in recent years as a result of improved access to affordable combination antiretroviral therapy (cART) drugs. Yet, access to services for prevention of common HIV-associated malignancies such as invasive cervical cancer (ICC) caused by carcinogenic human papillomavirus (HPV) remains inadequate. Thus, HIV-infected women remain at increased risk for HPV infection and cervical precancerous lesions  progressing to ICC. Very few studies have described HPV genotype composition among HIV-infected women in India. We conducted an observational study among HIV-infected women in Pune, India to expand the evidence base of HPV genotype distribution in this population.The study protocol was approved by the institutional ethical review boards of the National AIDS Research Institute, Pune, India and Vanderbilt University, Nashville, USA. All participants gave written, informed consent.The study was carried out in an outpatient gynecology clinic in a tertiary care hospital in Pune, India as part of the NIH-ICMR funded India-US HIV-Cervical Cancer Prevention Research Consortium. Consecutive women with documented serologic evidence of HIV infection were enrolled in the study. Participants were recruited regardless of their CD4+ cell counts or current status of receiving cART drugs. Exclusion criteria included a positive urine pregnancy test, debilitating illness that may preclude a pelvic examination, prior history of screening or treatment for cervical neoplasia, prior hysterectomy, and presence of current sexually transmitted infection.After explanation of study procedures and written informed consent, a structured questionnaire was administered to interview the participants and collect their sociodemographic information as well as key bio-behavioral risk factors relevant to HIV/AIDS and cervical cancer. Blood samples were obtained for CD4+ T-cell counts estimation . All enrolled women underwent a complete physical, pelvic, and colposcopic examination. Trained nurses collected cervical samples by spatula and cytobrush for conventional cervical cytology and HPV testing/genotyping. A standardized non-invasive colposcopy examination was performed on all participants by trained gynecologists. Confirmatory procedures for histology  were advised and performed only on consenting participants with clinical evidence of cervical abnormalities.and normal cytology), CIN1 , CIN2  and CIN3 (CIN3 on colposcopy/histology). This classification ensured that the most severe  cellular or tissue detection of dysplastic changes were included in the appropriate CIN disease status category.Cervical cytology and histopathology samples were analyzed by two experienced pathologists who reported diagnosis by consensus. The pathologists did not have knowledge of the HPV status of the participant. Cervical cytology results were reported as per revised (2001) Bethesda classification. We performed HPV genotyping on cervical specimens using PCR-based amplification of target DNA using the Linear Array® HPV genotyping test (LA-HPV) , an enhanced and commercialized version of the PGMY line blot assay (PGMY-LB) Data were analyzed using STATA intercooled version 10.0 and IBM SPSS Statistics 19. We analyzed prevalence of individual HPV genotypes (classified by carcinogenic risk categories and number of HPV genotypes per woman) in age categories, CD4+ cell strata, and cervical disease stages . Chi-square test for trend was used to analyze trends in proportion of women with prevalent HPV genotypes and CIN status.We fit bivariate and multivariable logistic regression models to identify associations between sociodemographic characteristics  and bio-behavioral factors  with HPV infection status. The dependent variables included (i) any prevalent HPV genotype, (ii) any prevalent ‘carcinogenic’ HPV genotypes, (iii) single carcinogenic HPV genotype, (iv) multiple carcinogenic HPV genotype, (v) HPV16 (the most common/most carcinogenic genotype), and (vi) any non-HPV16 carcinogenic type. We also evaluated the risk  of having high-grade cervical precancerous lesions (CIN3 and CIN2+) with the presence of individual carcinogenic HPV genotypes , with adjustment (as appropriate) for age, number of lifetime sexual partners, CD4+ cell counts, and presence of other carcinogenic HPV types. In an exploratory analysis, this risk of CIN3 and CIN2+ lesions was also estimated for combinations of individual carcinogenic HPV genotypes (single and multiple) with the concurrent presence of single and multiple non-carcinogenic types.A total of 278 HIV-infected women were enrolled for this study. The mean age was 32.3 years (S.D.: ±5.3), a third  were married and cohabiting with their husband, a third  were illiterate, and a majority  reported their family income of <2500 Indian Rupees per month (approximately US$55 at the time of the study). Half  of the participants reported age of first sexual intercourse as <18 years, while about one fifth  reported to have ≥2 lifetime sexual partners. Mean and median CD4+ cell counts were 411 /µL (S.D. ±214) and 372 /µL (interquartile range: 241–556) respectively.Cytology results revealed 7 women with HSIL, 41 with LSIL, 47 with ASC-US, 165 women with no squamous cell abnormality on cytology, while 18 women had inadequately stained smears. Colposcopic-histopathologic diagnoses revealed 13 women with CIN3, 16 women with CIN2, 46 women with CIN1, 193 women without any CIN abnormality, while colposcopy was unsatisfactory in 10 women. Three women had both unsatisfactory colposcopy and also cervical smears that were inadequately stained, such that the final composite cytologic-colposcopic-histopathological CIN disease status was able to be determined for 275 out of 278 women.The composite cytology-colposcopic-histopathological CIN diagnosis (N = 275) thus included no evidence of CIN in 143/275 (52%) women, CIN1 (CIN1/ASC-US/LSIL) in 101/275 (36.7%), CIN2 (CIN2/HSIL) in 18/275 (6.5%), and CIN3 in 13/275 (4.7%) women. Thus, the prevalence of CIN2+ was 11.2% (31/275) and that of CIN3 was 4.7% (13/275) in this population.At least one HPV genotype was detected in 146/278 (52.5%) participants, while multiple (≥2) HPV genotypes were present in 74/146 (50.7%) women. The number of HPV genotypes per woman ranged between 0–8, with a median of 1 genotype per woman. ‘Carcinogenic’ HPV genotypes were present in 98/278 (35.3%) women, ‘possibly carcinogenic’ genotypes in 41/278 (14.7%) while ‘non/unknown carcinogenic’ types were detected in 82/278 (29.5%) women. Out of 98 women with presence of any ‘carcinogenic’ HPV genotypes, 71/98 (72.4%) had a single ‘carcinogenic’ HPV genotype, 23/98 (23.4%) had two ‘carcinogenic’ HPV genotypes, while 4/98 (4.1%) had three ‘carcinogenic’ HPV genotypes, thus a total of 129 individual carcinogenic HPV infections were identified. Only 17/278 (6.1%) women with ‘possibly carcinogenic’ HPV genotypes and only 35/278 (12.6%) women with ‘non/unknown carcinogenic’ HPV genotypes were present without the concurrent presence of ‘carcinogenic’ HPV genotypes.The relative proportions of HPV infections (by carcinogenic risk categories) by age and CD4+ cell count categories are shown in Of the 37 HPV genotypes identifiable by the Linear Array assay, all except one (HPV69) were detected in the 278 samples. The commonest 10 genotypes were HPV16 (12.2%), HPV62 (7.3%), HPV71 (6.5%), HPV53 (6.2%), HPV42 (5.8%), HPV56 and HPV66 (both 4.4%), HPV18 and HPV39 (both 4%), and HPV35 and HPV51 (both 3.6%).  The carcAmong the 98 women with a total of 129 ‘carcinogenic’ HPV infections, HPV16 was the most common , although all 12 other ‘carcinogenic’ HPV genotypes were also present. (data not shown) Among women with presence of any single ‘carcinogenic’ HPV infection, as well as in women with evidence of CIN2+ and CIN3 lesions with single carcinogenic infections, HPV16 was still the commonest genotype, with an increasing proportion of 34%, 47%, and 50% respectively. Other ‘carcinogenic’ genotypes-HPV33, HPV39, HPV31, HPV56 and HPV35 were also present (in respectively decreasing fractions) in women with CIN2+ and CIN3 lesions with single carcinogenic HPV genotypes.At least one HPV genotype was present in 37.1% women with no CIN lesions, 66.3% in CIN1, 72.2% in CIN2 and 92.3% in CIN3 (p-for trend <0.001).  The trenWhen dichotomized at the high grade cervical neoplasia thresholds (CIN2+ and CIN3) thresholds of the cervical disease status, women with any HPV infection had higher risk for CIN2+ [Odds Ratio (OR) 4.31(95%CI: 1.71–10.87)] and CIN3 lesions [OR 11.64 (95%CI:1.49–90.80)] than women without HPV infection. Similarly, women with any ‘carcinogenic’ HPV genotypes had higher risk for CIN2+ [OR 5.51  and CIN2+ (vs. ≤CIN1) lesions , overall, as well as within cases of single carcinogenic HPV infections  and cases with presence of multiple carcinogenic infections . While other carcinogenic HPV genotypes had higher point estimates of Odds ratios than HPV16, their relative rarity led to wide 95% confidence intervals. When present as single carcinogenic HPV infections, HPV16, HPV31 and HPV33 had statistically significant higher risk of being present in CIN2+ while HPV16 and HPV33 had higher risk of CIN3. In cases with multiple carcinogenic infections, HPV16, HPV18, HPV56 and HPV58 had higher risk of being present in CIN2+ lesions while HPV58 and HPV68 had higher risk of being present in CIN3 lesions. An exploratory analysis of the risks of various single/multiple combinations of carcinogenic and non/unknown carcinogenic types is presented in In this cross sectional study, we have documented the diverse distribution of HPV genotypes and their associations with rigorously confirmed cervical precancerous disease endpoints, and factors associated with HPV infection among HIV-infected women in India. Overall, the prevalence of any HPV genotypes was 52.5% and prevalence of any ‘carcinogenic’ HPV genotypes was 35.3% in this cohort of HIV-infected women. The global estimates of HPV prevalence among HIV-infected women have varied by region and the level of the HIV epidemic. HPV16 was the commonest genotype (carcinogenic or otherwise) in our study in this population. It has been hypothesized HPV16 has better evolutionary ability to escape the effects of immune surveillance, while non-HPV16 genotypes are often better controlled by immune response. The overall diversity of HPV genotypes (regardless of carcinogenic grouping) found in our study is a characteristic uniformly reported from HIV-infected populations worldwide. This is in contrast to the substantially less diversity noted among HIV-uninfected women. Over half of our participants with HPV infection  had prevalent detection of multiple (≥2) HPV genotypes. The prevalence of multiple genotypes has varied widely (between 12%–87%) in similar studies worldwide We found that ≥2 lifetime sexual partners are associated with presence of any HPV infection, carcinogenic HPV infection, presence of multiple carcinogenic HPV types, and non-HPV16 carcinogenic types, but not with presence of single carcinogenic types, and with HPV16. This might be explained by the fact that HPV16  is highly transmittable in comparison with other carcinogenic types, thus its prevalent detection is regardless of the multiplicity of sexual partners. It was also noteworthy that other bio-behavioral factors  which are significant HPV co-factors in cervical carcinogenesis were not significantly associated with HPV infection status. However, we lacked adequate power to study these associations with HPV among women with high-grade cervical disease status. Elucidation of the independent or combined role of such cofactors affecting risk of carcinogenic HPV and incident cervical precancer and cancer will only be studied in prospective study designs.With the advantages of simultaneous detection of multiple genotypes, the LA-HPV assay also has certain limitations like cross-hybridization of the HPV52 probe with that of HPV33, HPV35, and HPV58 , lack of quantitation of HPV viral load, and chances of carry-over contamination. India has a high case burden of HIV/AIDS  as well as a high incidence of ICC . Table S1Relationship of prevalent carcinogenic and concurrent non/unknown-carcinogenic types (single or multiple) with risk of CIN2+ and CIN3 in HIV-infected women.(DOC)Click here for additional data file.


Although G6PDd individuals are generally asymptomatic throughout their life, the clinical burden of this genetic condition includes a range of haematological conditions, including acute haemolytic anaemia (AHA), neonatal jaundice (NNJ) and chronic non-sphaerocytic anaemia (CNSA). In Latin America (LA), the huge knowledge gap regarding G6PDd is related to the scarce understanding of the burden of clinical manifestation underlying G6PDd carriage. The aim of this work was to study the clinical significance of G6PDd in LA and the Caribbean region through a systematic review.A systematic search of the published literature was undertaken in August 2013. Bibliographies of manuscripts were also searched and additional references were identified. Only original research was included. All study designs were included, as long as any clinical information was present. Studies were eligible for inclusion if they reported clinical information from populations living in LA or Caribbean countries or about migrants from these countries living in countries outside this continent.P. vivax malaria patients during the course of the treatment with primaquine (PQ). Favism, infection-induced haemolysis, NNJ and CNSA appear to play only a minor public health role in this continent.The Medline search generated 487 papers, and the LILACS search identified 140 papers. After applying the inclusion criteria, 100 original papers with any clinical information on G6PDd in LA were retrieved. Additionally, 16 articles were included after reading the references from these papers. These 116 articles reported data from 18 LA and Caribbean countries. The major clinical manifestations reported from LA countries were those related to AHA, namely drug-induced haemolysis. Most of the published works regarding drug-induced haemolysis in LA referred to haemolytic crises in P. vivax infection in this continent, although the low number of reported cases, which could be linked to under-reporting of complications. These results support the need for better strategies to diagnose and manage G6PDd in malaria field conditions. Additionally, Malaria Control Programmes in LA should not overlook this condition in their national guidelines.Haemolysis in patients using PQ seems to be the major clinical manifestation of G6PDd in LA and contributes to the morbidity of  Plasmodium falciparum malaria [circa 350 million affected individuals in these countries. The lowest prevalence is known to occur in the Americas, while the highest prevalence is observed in tropical Africa, Middle East and the Mediterranean basin [Glucose-6-phosphate dehydrogenase (G6PD) is an important enzyme that catalyses the first reaction in the pentose-phosphate pathway. Within the erythrocyte, G6PD is the sole source of enzymatic activity that protects against the build-up of super-radicals and, thus, oxidative stress ,2. G6PD  malaria ,4. TherePlasmodium vivax infections, as radical cures, including the specific treatment of the latent hepatic stages that are mainly responsible for subsequent relapses after the first clinical episode, require the use of PQ for 7 to 14 days. In some individuals, this treatment can lead to life-threatening anaemia and acute renal failure [P. falciparum stage V gametocytes. The potentially hazardous effect of this drug in G6PDd individuals jeopardizes any population-wide effort to reduce malaria transmission via drugs such as PQ due to the impossibility of performing rapid population-wide screening for the deficiency and the hazards associated with the blind administration of such drugs [P. falciparum, as this dose is efficacious as a gametocytocidal drug without the risks of inducing haemolysis in G6PD patients [Although G6PDd individuals are generally asymptomatic throughout their life, the clinical burden of this genetic condition includes a range of haematological conditions, including acute haemolytic anaemia (AHA), neonatal jaundice (NNJ) and chronic non-sphaerocytic anaemia (CNSA) ,2. Becaupatients .A thorough understanding of the clinical burden related to G6PDd could be of great value for understanding whether particular drug regimens could be used safely at a population level with or without prior knowledge of the individuals’ G6PD status, or whether the development of field-deployable and electricity-independent rapid diagnostic tests, resembling those that already exist for malaria diagnosis, is indispensable. Some authors have recently proposed maps of the G6PDd global distribution, including LA countries, highlighting the small amount of published works regarding this deficiency in this continent ,9. Large2, which included a population of approximately 580 million inhabitants in 2008. With regards to ethnicity, in some countries, mainly Chile, Argentina and Uruguay, white people of European origin predominate. In others, such as Venezuela, Brazil, Paraguay, Nicaragua and Colombia, white immigrants mixed noticeably with the Amerindians. Finally, in some countries, such as Mexico, Guatemala, El Salvador, Honduras, Panama, Peru, Bolivia and Ecuador, the Amerindians still constitute a numerically important portion of the population. Additionally, the continent has received multiple waves of African immigrants, especially to the Caribbean area.South America, Central America, Mexico and the Caribbean area constitute a vast territory formed by 33 independent countries and 20 autonomous or semi-autonomous territories. They cover a territorial area of over 20 million kmPotentially relevant papers in all languages were accessed from Medline and LILACS to review their full texts. A broad free text search using the combination of Medical Subject Heading (MeSH) terms and keywords presented in Table proxy for AHA. To identify relevant papers, the titles, abstracts and the full texts of the studies were examined by two independent reviewers. The data were directly extracted from the full-length articles to structured table and figures containing all the descriptive variables and relevant outcomes.Only original research of all study designs  was included, as long as any clinical information was present. Additional articles were obtained through citation tracking of reviews, opinion articles and original papers. Studies were eligible for inclusion if they reported clinical information from populations living in LA or Caribbean countries or about migrants from these countries living in countries outside this continent. An individual was considered to have G6PDd if they presented with any positive diagnostic test, such as DNA analysis, enzyme activity assay, NADPH fluorescent spot, brilliant cresyl blue, gel electrophoresis, methylene reduction test or phenazine methosulphate-3--2,5-diphenyltetrazolim bromide. In this study, clinical information refers to G6PDd manifestations, such as AHA, NNJ and CNSA, as well as G6PDd coexisting with other genetic disorders and association of G6PDd with susceptibility or clinical presentations of metabolic disorders or other infectious diseases than malaria. In this study, a history of jaundice or red cell transfusion after drug therapy was used as a The Medline search generated 487 papers, and the LILACS search generated 140 papers. After applying the inclusion criteria to those papers, 100 original papers with any clinical information on G6PDd in LA were retrieved. Additionally, 16 articles were included after reading the references of the 100 articles and reviews/ opinion articles that were obtained from the Medline and LILACS searches  subjects, being 8 (17.0%) presenting PQ-induced haemolysis, 8 with haemolysis induced by other drugs/substances (44.4%), 6 with favism (66.7%) and 2 (66.7%) with infection-induced haemolysis. PQ-induced haemolysis cases predominated among admixed individuals . Haemolysis induced by other drugs/substances  and infection-induced haemolysis  predominated among black individuals. Favism was recorded in white individuals only .Figure Some cross-sectional studies with a recall approach showed an absence of signs of haemolysis among the G6PD deficient population -14. One P. falciparum gametocytes. In Mexico [S. mansoni were given a single intramuscular dose of hycanthone, but their subsequent serial haematocrit and reticulocyte counts showed no evidence of haemolysis [Reports of occasional moderate haemolytic anaemia were reported in Brazil , Costa Rn Mexico , Ecuadorn Mexico  and Chiln Mexico , the fren Mexico . In LA cn Mexico , chloramn Mexico , aspirinn Mexico ,29, sulpn Mexico ,30,31, sn Mexico  and acetn Mexico . In Brazn Mexico , ketoconn Mexico , or who n Mexico . In Sainemolysis .P. vivax infection could only attribute the cause of death to PQ-induced haemolysis, demonstrating the lethal potential of this condition [In 1970, three patients from El Salvador who presented with PQ-induced haemolysis were confirmed as G6PD deficient, and one of these patients required an exchange transfusion . Two casondition .The use of chloroquine alone did not trigger haemolysis in 8 G6PD-deficient subjects .The search identified five publications from Chile that reported a total of nine cases of favism in LA ,46-49. AIn Cuba, recurrent viral and bacterial infections were indicated as possible causes of haemolysis in an 8-year-old boy . In CuraNeonatal jaundice was reported in 30 original articles. The frequency of NNJ in G6PD-deficient new-borns ranged from 38.5 to 100% -56. HoweSome cross-sectional studies have demonstrated that NNJ cannot be solely attributed to G6PDd  or a lacData concerning the association between G6PDd and anaemia, including chronic non-sphaerocytic anaemia, were presented in 18 articles. Only 8 defined cases of CNSA were recorded from Latin America, with 2 from Brazil ,79, 2 frMost of the published reports describing haematological parameters in LA, which covered several distinct geographical areas, showed similar haematimetric indexes in G6PDd individuals and non-deficient ones -92. In BThe coexistence of G6PDd with other genetic disorders was reported in 14 articles. In Argentina, beta-thalassemia was detected in 4.8% of the G6PD-deficient subjects , and thePlasmodium falciparum infections were recorded at the same proportion in G6PDd and normal individuals in the Southern Brazilian Amazon [P. falciparum[P. falciparum parasitaemia than persons without this condition [P. vivax malaria than in those presenting with the disease [P. vivax predominates, G6PDd individuals were less likely to report the occurrence of malaria episodes after adjusting for age [An association between G6PDd and malaria susceptibility was reported in 8 articles. In a traditionally malaria-endemic area of South-eastern Brazil, the frequency of G6PDd and average G6PD activity was similar between the groups with and without a history of malaria . Plasmodn Amazon . In Brazn Amazon . Howeveralciparum. Colombiondition . In Colo disease . In the  for age ,43. In tThe presence of metabolic disorders in G6PDd patients was investigated in three articles. Significantly lower insulin levels were observed for G6PDd Brazilian men compared to the controls in both the intravenous and oral tolerance tests . In MexiP. vivax presenting G6PDd [Loxosceles[S. mansoni[Other findings were mentioned in 10 original articles. A significant increase in methaemoglobinaemia was observed following oral therapy with PQ in patients with ng G6PDd ,116. Addng G6PDd . In Brazoxosceles. In Brazoxosceles,120. In oxosceles. Transfuoxosceles. In Braz. mansoni and lepr. mansoni.P. vivax is more widely distributed than P. falciparum and that this species is a potential cause of morbidity and mortality amongst the 2.85 billion people living at risk of infection, most of whom live in Central and South East Asia and LA [P. vivax infections requires the use of PQ, which can lead to acute intravascular haemolysis in G6PDd individuals, resulting in severe anaemia and acute renal failure [Mapping studies have demonstrated that a and LA . In LA aa and LA . The pre failure . DespiteThe most commonly reported clinical manifestations reported from LA countries were those related to AHA, namely drug-induced haemolysis. In patients with haemolytic anaemia, the reported frequency of G6PDd could be higher than 30% ,26,126. P. vivax malaria during the course of the treatment with PQ. Reports of the need for red cell transfusions were common in these cases. In this continent, complications such as severe anaemia and renal failure also seem to be common and are of public health concern. Likewise, lethality was reported in the Brazilian Amazon, highlighting the impact on the local public health systems. Most strikingly, the results of two studies showed that the frequency of haemolysis in G6PDd patients was almost 90% [P. vivax.Most of the published works regarding drug-induced haemolysis in LA referred to haemolytic crises in patients diagnosed with most 90% ,39. SpecAmerican countries contribute to 4.5% of the G6PDd male population from malaria endemic countries, corresponding to an estimated 10 million males, according to population data from 2010 [This review points to the need for systematic testing of G6PDd in LA malaria endemic countries. Only with this result would it be possible to appropriately guide treatment, adjusting the PQ dosage or even contraindicating the drug, depending on the enzymatic activity of G6PD. The exposed population is extremely large in many endemic areas, and testing for G6PD status in all individuals would be a costly exercise, the cost-effectiveness of which remains to be evaluated. Furthermore, only a small number of investigations estimating G6PDd prevalence and the relative frequencies of the different genetic variants were carried out in LA malaria-endemic areas. Studies developed in the Brazilian Amazon found an estimated prevalence of 4%, with the African and Mediterranean variants predominating . This reP. vivax malaria endemic areas, and consequently the areas where PQ is prescribed, namely the Amazon. Favism was recorded in white individuals, in agreement with the literature that points that this is a condition typical of males of Mediterranean descent.A sound analysis on the role of the ethnicity in the geospatial distribution of G6PDd-related events was greatly impaired by the lack of information on ethnicity background for research subjects. It is known that global prevalence of G6PDd, and probably of its clinical manifestations, is geographically correlated with areas inhabited by populations historically exposed to endemic malaria, including Africa, Middle East, Mediterranean Europe and South-East Asia . In thisAs previously demonstrated, the combination of G6PDd with the sickle cell trait was no more frequent than that expected by chance . SeveralP. falciparum infections, it has been demonstrated that the shorter half-life and rapid clearance of red cells of G6PDd individuals make them less susceptible to malarial attacks from these parasites [P. vivax-infected cells. However, there is a need to further explore and comprehend the mechanisms by which individuals with G6PDd are rendered less susceptible to infection. P. vivax preferentially invades reticulocytes, but P. falciparum can invade erythrocytes of all ages. This erythrocyte invasion is a significant phenomenon because G6PD activity is markedly reduced in older erythrocytes. Thus, red cells infected with P. vivax are most likely less vulnerable to the oxidant stress produced by the parasite. The precise mechanism by which G6PDd promotes reduced susceptibility to vivax malaria remains to be established.In LA, there are results suggesting that G6PDd prevalence could be a marker of the selective pressure exerted by malaria. The geographic distribution of G6PDd suggests that some polymorphisms confer resistance to falciparum malaria . This pharasites , and it P. vivax hypnozoites is PQ. Tafenoquine is another drug of the same group that is being evaluated in clinical trials [P. vivax in LA, thus representing the major clinical complication of G6PDd in this continent. PQ-induced haemolysis was only reported in males, thus indicating that testing only this population for G6PDd may be a more efficient and cheaper measure for minimising the risk of clinically relevant haemolysis after PQ administration in LA malaria endemic areas.Currently, the only drug available for the elimination of l trials . This unIt is likely that the real impact of G6PDd in terms of malaria-related complications has been heavily underestimated, and further research should be devoted to clarifying the real burden that these complications impose on the health systems. Finally, this study highlights the need to improve current strategies for diagnosing and managing G6PDd in malaria field conditions. Malaria control programmes in LA need to take this condition into serious account in their national guidelines if measures such as massive PQ administration are considered as part of regional malaria elimination agendas.AHA: Acute haemolytic anaemia; CHSA: Chronic non-sphaerocytic anaemia; G6PD: Glucose-6-phosphate dehydrogenase; G6PDd: Glucose-6-phosphate dehydrogenase deficiency; LA: Latin America; NNJ: Neonatal jaundice; PQ: Primaquine.MVGL was one of the principal investigators of the multicentre tafenoquine phase II trial, which is funded by GSK. The other authors declare that they have no competing interests.WMM and MVGL conceived and designed the study. WMM and GPF performed a systematic review of the primary literature. WMM, GPF, ALMQ and GCM drafted the manuscript. MB, MRFO, HP, GASR. QB and MVGL directed the organisation and edited the manuscript. All authors read and approved the final version of the manuscript.Summary of the findings organized by the type of stressor triggering haemolysis in G6PDd patients.Click here for file